Co-reporter:Shuiqin Jiang;Lujia Zhang;Zhiqiang Yao;Bei Gao;Hualei Wang;Xiangzhao Mao
Catalysis Science & Technology (2011-Present) 2017 vol. 7(Issue 5) pp:1122-1128
Publication Date(Web):2017/03/06
DOI:10.1039/C7CY00060J
The development of robust biocatalysts producing a large range of organic amides by hydration of nitriles is an important pursuit and challenge. A nitrilase with a broad range of nitrile substrates was switched to a nitrile hydratase by rationally regulating the reaction pathways. Five mutants improved the amide formation in the product, and four of them formed >50% amide. F193N, with the highest amide formation among the four mutants, improved its amide product up to 73%, which was 35-fold that of the wild type, while maintaining 50% activity relative to the wild type. This study would afford a new synthetic route to amides from nitriles and could be a valuable addition to the synthetic repertoire. Further protein engineering may expand the reaction range of an enzyme to afford more additional pathways to synthetic biology.
Co-reporter:Xinyi Tao, Ning Jia, Nenghui Cheng, Yuhong Ren, Xuni Cao, Min Liu, Dongzhi Wei, Feng-Qing Wang
Biomaterials 2017 Volume 131(Volume 131) pp:
Publication Date(Web):1 July 2017
DOI:10.1016/j.biomaterials.2017.03.045
•A novel strategy to develop self-assembled phosphatidyl prodrug catalyzed with PLD.•Targeted drug release of phosphatidyl drug in tumor cells triggered by overexpressed PLD.•High drug loading capacity and stability of phosphatidyl prodrug.•Reduced toxicity and changed biodistribution resulted from the phosphatidyl modification of drug.•Reversed effect of phosphatidyl prodrug on multidrug resistance cell.A strategy is proposed to design a safe and simple amphiphilic prodrug delivery system, based on the elevated expression of phospholipase D (PLD) in cancer cells. The method utilizes the transphosphatidylation ability of bacterial PLD on alcohol groups and the hydrolysis activity of overexpressed PLD on phospholipids in cancer cells. Doxorubicin (DOX) was selected as a test drug, and the phosphatidyl-doxorubicin (PX) was synthesized by bacterial PLD. The PX prodrug could be readily self-assembled to nanoparticles with uniform size and was stable during storage and circulation. The pharmacokinetics and biodistribution investigations indicated DOX could be selectively released from PX in cancer cells triggered by the local overexpressed PLD, and PX could significantly prolong the half-life of DOX in the tumors and decrease the distribution in heart and kidney. Moreover, the PX prodrug enhanced cellular uptake in MCF-7/ADR cells, demonstrating it could reverse the multi-drug resistance. Consequently, the prodrug displayed favorable anticancer efficacy in the MCF-7/ADR xenograft model without the cardiotoxicity and nephrotoxicity of DOX. The results demonstrated that phosphatidyl modification method can be used as an efficient strategy to develop a promising nanoscale drug delivery system for some drugs.
Co-reporter:Wei Wang, Yumeng Chen, Dong-Zhi Wei
Journal of Microbiological Methods 2017 Volume 143(Volume 143) pp:
Publication Date(Web):1 December 2017
DOI:10.1016/j.mimet.2017.10.006
•We describe copper-mediated on-off control of gene expression in Trichoderma reesei.•We established a copper-mediated unmarked genetic modification strategy.•Our copper-mediated system is useful in controlling gene expression in T. reesei.We developed a copper-mediated gene expression on-off control system and an unmarked genetic modification strategy based on the modified copper responsive promoter Ptcu1c from the filamentous fungus Trichoderma reesei. The molecular tool for the gene expression on-off control can be used to manipulate many biological processes in T. reesei.Download high-res image (131KB)Download full-size image
Co-reporter:Mingrong Guo, Xujun Chen, Yanling Wu, Lujia Zhang, Weixue Huang, Ying Yuan, Ming Fang, Jingli Xie, Dongzhi Wei
Process Biochemistry 2017 Volume 63(Volume 63) pp:
Publication Date(Web):1 December 2017
DOI:10.1016/j.procbio.2017.08.009
•Three novel ACE inhibitory peptides were isolated from P. esculenta.•Three peptides inhibit ACE in non-competitive manner.•Molecular docking was applied for the illustration of inhibitory mechanism.•Peptides YASGR and GNGSGYVSR significantly decrease the blood pressure of SHRs.Three novel angiotensin-I converting enzyme inhibitory peptides were explored from Sipuncula (Phascolosoma esculenta), a seafood with high protein content. Peptides RYDF, YASGR and GNGSGYVSR were obtained by hydrolysis of the water-soluble protein of Sipuncula with pepsin and trypsin. The peptides were purified through gel filtration and reverse-phase high-performance liquid chromatography, and identified by de novo sequencing method of MALDI-TOF. All three peptides are non-competitive inhibitors of angiotensin-I converting enzyme determined by Lineweaver-Burk plots. Their inhibitory IC50 values were 235, 184 and 29 μM, respectively. The inhibitory mechanism was well illustrated through molecular docking. The docking results showed that the differences of inhibitory activities of the three peptides were due to the degree of non-covalent bond-based interactions between the peptides and angiotensin-I converting enzyme, especially the hydrogen bonds. The antihypertensive effect of peptides was confirmed by their lowering blood pressure in spontaneously hypertensive rats with the oral administration as 5 mg/kg body weight. Peptide GNGSGYVSR decreased systolic blood pressure 31 mmHg at 2 h after oral administration, and maintained the level till 4 h. Therefore, peptides from Sipuncula can be considered as promising candidates for ACE inhibition and hypertension treatment.Download high-res image (206KB)Download full-size image
Co-reporter:Zhiqiang Yao, Lujia Zhang, Bei Gao, Dongbing Cui, Fengqing Wang, Xiao He, John Z. H. Zhang, and Dongzhi Wei
Journal of Chemical Information and Modeling 2016 Volume 56(Issue 10) pp:1979-1994
Publication Date(Web):August 16, 2016
DOI:10.1021/acs.jcim.5b00585
The discovery of unique substrates is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming, and expensive. Although in silico structure-based approaches show great promise, recent extensive studies have shown that these approaches remain a formidable challenge for current biocomputational methodologies. Here we present an open-source, extensible, and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of an enzyme on the basis of the enzyme’s catalysis mechanism. On the basis of a generalized transition state theory of enzyme catalysis, we introduce a modified docking procedure called “mechanism-based restricted docking” (MBRD) for novel substrate recognition from molecular docking. Comprising a series of utilities written in C/Python, THEMIS automatically executes parallel-computing MBRD tasks and evaluates the results with various molecular mechanics (MM) criteria such as energy, distance, angle, and dihedral angle to help identify desired substrates. Exhaustive sampling and statistical measures were used to improve the robustness and reproducibility of the method. We used Candida antarctica lipase B (CALB) as a test system to demonstrate the effectiveness of our computational prediction of (non)substrates. A novel MM score function for CALB substrate identification derived from the near-attack conformation was used to evaluate the possibility of chemical transformation. A highly positive rate of 93.4% was achieved from a CALB substrate library with 61 known substrates and 35 nonsubstrates, and the screening rate has reached 103 compounds/day (96 CPU cores, 100 samples/compound). The performance shows that the present method is perhaps the first reported scheme to meet the requirement for practical applicability to enzyme studies. An additional study was performed to validate the universality of our method. In this verification we employed two distinct enzymes, nitrilase Nit6803 and SDR Gox2181, where the correct rates of both enzymes exceeded 90%. The source code used will be released under the GNU General Public License (GPLv3) and will be free to download. We believe that the present method will provide new insights into enzyme research and accelerate the development of novel enzyme applications.
Co-reporter:Kai Wu, Lifeng Chen, Haiyang Fan, Zhiqiang Zhao, Hualei Wang, Dongzhi Wei
Tetrahedron Letters 2016 Volume 57(Issue 8) pp:899-904
Publication Date(Web):24 February 2016
DOI:10.1016/j.tetlet.2016.01.048
Enantiopure α-phenethyl alcohols, including aromatic halohydrins, are important chiral building blocks. One of the best approaches to synthesise α-phenethyl alcohols is asymmetric reduction of prochiral ketones by alcohol dehydrogenases (ADHs). The obtained enantiopure halohydrin could be directly used to produce enantiopure epoxide through a base-induced ring-closure reaction, which is an attractive ‘one pot’ chemoenzymatic method for producing high-yield epoxide. In this study, a novel medium-chain dehydrogenase (KcDH) from Kuraishia capsulate CBS1993 was identified and characterised to show its broad substrate scope and excellent enantioselectivity. KcDH showed activities on 25 substrates of the 26 tested aromatic ketones and heteroaryl ketones, with an enantiomeric excess (ee) >99% and the highest relative activity observed with para-nitro acetophenone. Due to its high enantioselectivity for α-haloketones, a chemoenzymatic method for the synthesis of enantiopure styrene oxide (SO) and phenyl glycidyl ether (PGE) was developed through a base-induced ring-closure reaction on enantiopure halohydrin obtained with KcDH. (R)-SO and (S)-PGE were obtained in 86% and 94% analytical yield, respectively, and both epoxides were obtained with ee >99%. Thus, our results suggested that KcDH may be a promising biocatalyst for the production of multiple enantiopure α-phenethyl alcohols and epoxides.
Co-reporter:Xi-hua Zhao;Wei Wang;Bin Tong;Su-ping Zhang
Applied Biochemistry and Biotechnology 2016 Volume 178( Issue 1) pp:173-183
Publication Date(Web):2016 January
DOI:10.1007/s12010-015-1866-x
Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher β-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of β-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50–60 °C and 4–5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.
Co-reporter:Min Liu;Zhan-Tao Zhu;Xin-Yi Tao;Feng-Qing Wang;Dong-Zhi Wei
Biotechnology Letters 2016 Volume 38( Issue 11) pp:1881-1892
Publication Date(Web):2016 November
DOI:10.1007/s10529-016-2187-z
To investigate single nucleotide polymorphism (SNP) in the transformation process of phytosterol to valuable steroid intermediates in three steroid-producing Mycobacterium neoaurum strains using deep sequencing and bioinformation analysis.The assembled contig sequences from RNA sequencing of strains producing 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA) were analyzed for the presence of putative SNPs for steroid catabolism. 413, 375, and 491 SNPs were detected in the coding domain sequences and non-coding domain sequences of RNA sequencing reads of M. neoaurum strains producing 9OHAD, ADD, and BNA, respectively. Special attention was focused on SNPs associated with genes showing differential expression at proteome level, including the genes for sterol catabolism, glycerol catabolic process, signal transduction systems, transport system and energy metabolism.The work facilitates the understanding of underlying genetic changes that may be responsible for steroid accumulation in M. neoaurum and is useful for its targeted genetic engineering.
Co-reporter:Yujie Chen;Dong Yi;Shuiqin Jiang
Applied Microbiology and Biotechnology 2016 Volume 100( Issue 7) pp:3101-3111
Publication Date(Web):2016 April
DOI:10.1007/s00253-015-7129-5
ω-Transaminases (ω-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified ω-TAs is important to broaden their potential for synthetic application. Taurine–pyruvate TA (TPTA, EC 2.6.1.77) is an ω-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTAgth) and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 9.0 and 65 °C, with remarkable thermostability and tolerance toward organic solvents. Its KM and vmax values for taurine were 5.3 mM and 0.28 μmol s−1 mg−1, respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTAgth was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTAgth were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTAgth showed a unique mechanism of sulfonate/α-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.
Co-reporter:Kai Wu;Hualei Wang;Lifeng Chen;Haiyang Fan
Applied Microbiology and Biotechnology 2016 Volume 100( Issue 20) pp:8757-8767
Publication Date(Web):2016 October
DOI:10.1007/s00253-016-7631-4
Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive “1-pot, 2-step” chemoenzymatic approach for producing enantiopure SO with 100 % theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1 % by adding methyl tert-butyl ether (MTBE) as the second phase. Therefore, with this optimized approach, ADH from Lactobacillus kefir (LkDH) successfully produced 1 M (S)-SO, with 99 % analytical yield and 97.8 % enantiomeric excess (ee). In the preparation of (R)-SO, a semi-rational strategy of active pocket iterative saturation mutagenesis (ISM) was successfully used to inverse the enantioselectivity of LkDH (muDH2, F147L/Y190P/A202F/M206H/V196L/S96D/K97V), which produced the opposite enantiomer (R)-2-chloro-1-phenylethanol. Through the optimized chemoenzymatic approach, muDH2 was successfully used to prepare 1 M (R)-SO, with 98.1 % ee and 99.0 % analytical yield. Our results indicated that this optimized chemoenzymatic approach could be used to produce both enantiomers of SO at concentrations as high as 120 g/L within 14 h, which is the highest concentration as far as we know. MuDH2 obtained through ISM also showed reversed enantioselectivity toward another 13 aromatic ketones, compared with wild-type (WT) LkDH. Furthermore, a molecular docking experiment demonstrated that muDH2 inverted the binding orientation of the substrate, which may be the reason for its inverse enantioselectivity.
Co-reporter:Shujing Zhu, Xiaoqiang Ma, Erzheng Su and Dongzhi Wei
Green Chemistry 2015 vol. 17(Issue 7) pp:3992-3999
Publication Date(Web):02 Jun 2015
DOI:10.1039/C5GC00169B
From an environmental perspective, utilizing nonconventional solvents (i.e. green solvents) with low ecological footprints is a highly beneficial alternative to using conventional organic solvents to form a reaction system. The nitrile hydratase (NHase, EC 4.2.1.84) catalyzed hydration of 2-amino-2,3-dimethylbutyronitrile (ADBN) to 2-amino-2,3-dimethylbutyramide (ADBA) in various green solvent–aqueous reaction systems was investigated in this study. After systematically optimizing the reaction conditions, the HFE-7100/H2O (v/v, 10%) biphasic system was ultimately identified as a promising reaction system for reducing product inhibition, avoiding substrate hydrolysis, and facilitating product separation and solvent recovery. The average ADBA yield of an entire batch reaction was 97.3%, which is obviously higher than those obtained with previously reported chemical or enzymatic methods. This is the first attempt to apply a fluorous solvent–aqueous biphasic system to a biocatalytic process, and the results suggest that the fluorous solvent employed in the biphasic system satisfies the requirements for green chemistry.
Co-reporter:Xin Gao, Chengcheng Zhao, Ting Yu, Shengli Yang, Yuhong Ren and Dongzhi Wei
Chemical Communications 2015 vol. 51(Issue 50) pp:10131-10133
Publication Date(Web):14 May 2015
DOI:10.1039/C5CC02544C
A strategy for constructing a reusable multi-enzyme supramolecular device was developed by reprogramming protein–protein interactions and disulfide bond locking. The resultant multi-enzyme supramolecular device demonstrated good reusability, and approximately 80% of its initial catalytic activity was retained even after eight cycles of reuse.
Co-reporter:Hualei Wang, Haiyang Fan, Huihui Sun, Li Zhao, and Dongzhi Wei
Organic Process Research & Development 2015 Volume 19(Issue 12) pp:2012-2016
Publication Date(Web):October 15, 2015
DOI:10.1021/acs.oprd.5b00269
(R)-(−)-Mandelic acid is an important chiral building block that is widely used in pharmacy and the production of fine chemicals. A more advanced method for obtaining (R)-(−)-mandelic acid is direct hydrolysis of the corresponding racemic mandelonitrile. In order to develop a cost-effective process, a highly efficient enantioselective nitrilase BCJ2315 from Burkholderia cenocepacia J2315 was used for the biotransformation of mandelonitrile to (R)-(−)-mandelic acid. The recombinant Escherichia coli M15/BCJ2315 showed high substrate tolerance and could completely hydrolyze up to 250 mM of mandelonitrile. A fed-batch reaction was performed by periodically or continuously dosing the substrate into the reactor to alleviate substrate inhibition in a monophasic buffer system. Finally, the highest substrate loading (2.9 M) was achieved in the continuous fed batch reaction mode, giving (R)-(−)-mandelic acid at the highest concentration (2.3 M, 350 g/L) with 97.4% ee ever reported. The hydrolysis process was easily scaled up to 2 and 10 L, indicating the potential for the industrial production of optically pure (R)-(−)-mandelic acid.
Co-reporter:Kai Wu;Hualei Wang;Huihui Sun
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 22) pp:9511-9521
Publication Date(Web):2015 November
DOI:10.1007/s00253-015-6716-9
Enantioselective hydrolysis of racemic epoxides mediated by epoxide hydrolases (EHs) is one of the most promising approaches to obtain enantiopure epoxides. In this study, we identified and characterized a novel EH (TpEH1) from Tsukamurella paurometabola by analyzing the conserved catalytic residues of EH. TpEH1 was overexpressed and purified, and its catalytic properties were studied using racemic phenyl glycidyl ether (PGE) and its derivatives as substrates. TpEH1 showed excellent enantioselectivity to the substrates PGE, 3-methylPGE, and 3-nitroPGE. The highest enantioselectivity (E > 100) was achieved when 3-nitroPGE was used as the substrate. The recombinant Escherichia coli TpEH1 demonstrated high substrate tolerance toward PGE and could hydrolyze PGE at concentrations of up to 400 mM (60 g/L) with high enantioselectivity (E = 65), giving (R)-PGE with enantiomeric excess of more than 99 % ee and 45 % yield within 1 h. This concentration of PGE is the highest reported concentration catalyzed by native EHs to date. Thus, the easily available and highly active E. coli TpEH1 showed great potential for the practical preparation of optically pure (R)-PGE.
Co-reporter:Xiao-qiang Ma;Er-zheng Su;Sen-wen Deng
Biotechnology and Bioprocess Engineering 2015 Volume 20( Issue 4) pp:718-724
Publication Date(Web):2015 August
DOI:10.1007/s12257-013-0607-7
In this study, several chemical treatment techniques commonly used for protein extraction were investigated for recovering glutaryl-7-aminocephalosporanic acid acylase (GLA) from recombinant E. coli cells. The best results were obtained by the combined use of cetyltrimethylammonium bromide (CTAB) and KCl. Subsequently, various extraction conditions, such as cation salt, concentrations of CTAB and KCl, extraction temperature, extraction time, and biomass, were optimized to further enhance the release yield and specific activity of GLA. Our results showed that 110% of GLA was released after treatment with 0.5% CTAB (w/v, %) and 0.3 M KCl at 10°C for 12 h, and its specific activity in this extracting solution was approximately 1.5 times higher as compared to that obtained by sonication. This extraction method could avoid the inactivation of GLA caused by drastic mechanical methods, and also enhance its specific activity for industrial extraction.
Co-reporter:Li Zhao;Jinping Lin;Hualei Wang;Jingli Xie
Bioprocess and Biosystems Engineering 2015 Volume 38( Issue 12) pp:2487-2495
Publication Date(Web):2015 December
DOI:10.1007/s00449-015-1486-4
In this work, a two-step process was developed for the production of 3-hydroxypropionic acid from glycerol. In the first step, glycerol was converted to 1,3-propanediol by Klebsiella pneumonia. In the second step, the 1,3-propanediol was converted into 3-hydroxypropionic acid by Gluconobacter oxydans. In a 7.0 L bioreactor, the whole process took 54 h, consumed 480 g glycerol and produced 242 g 3-hydroxypropionic acid. The conversion rate of glycerol to 3-hydroxypropionic acid was 50.4 % (g g−1). The final concentration of 3-hydroxypropionic acid arrived 60.5 g L−1. The process was effective for 3-HP production from glycerol and it might provide a new approach to the biosynthesis of 3-HP from a cheap starting material. Moreover, in this paper, it was first reported that the by-product of 3-hydroxypropionic acid production from 1,3-propandeiol was acrylic acid.
Co-reporter:Jian Zhang;Xinxin Lv;Rui Xu;Xinyi Tao
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 16) pp:6705-6713
Publication Date(Web):2015 August
DOI:10.1007/s00253-015-6441-4
Interleukin-24 (IL-24), a cytokine belonging to the IL-10 family, can selectively induce apoptosis in a broad range of tumor cells without harming normal cells. The efficient and soluble expression of bioactive recombinant IL-24 in Escherichia coli remains an obstacle because of aggregation and insufficient yield. In this study, a fusion of the small ubiquitin-related modifier (SUMO) or maltose-binding protein (MBP) has shown potential in facilitating the produce of IL-24. Thus, a new construct for MBP-SUMO-IL-24 expression would be a promising approach. Our results showed that the MBP-SUMO-IL-24 fusion protein was efficiently expressed as a soluble protein. SUMO protease-mediated cleavage at the SUMO/IL-24 junction released the recombinant IL-24 from the fusion protein. In addition, a His6 tag fused upstream of SUMO allowed for one-step purification through nickel affinity chromatography. Cleavage of the MBP-SUMO tag on the column resulted in the release of purified IL-24 and simplified the purification process. The final yield of IL-24 with approximately 90 % purity was 19 mg/L in flask fermentation. In vitro activity assays demonstrated that the purified IL-24 could induce apoptosis in MCF-7 breast cancer cells, but not normal NHLF cells, in a dose-dependent manner. In summary, we developed a novel method to express soluble and bioactive IL-24 protein in prokaryotic cells.
Co-reporter:Hualei Wang, Huihui Sun, Wenyuan Gao, and Dongzhi Wei
Organic Process Research & Development 2014 Volume 18(Issue 6) pp:767-773
Publication Date(Web):September 3, 2013
DOI:10.1021/op400174a
A solvent engineering approach and an extended fed-batch reaction mode were introduced to increase the activity and enantioselectivity and alleviate the substrate inhibition of nitrilase BCJ2315 from Burkholderia cenocepacia J2315 toward o-chloromandelonitrile. Among the seven water-miscible organic solvents tested, ethanol (30%, v/v) demonstrated the highest reaction conversion (55.7%) and enantioselectivity (enantiomeric excess, 98.2% ee) compared with those of the control [which did not contain any organic solvent (13% and 89.2%, respectively)] and was thus chosen as the suitable cosolvent. In the extended fed-batch reaction mode, o-chloromandelonitrile (solubilized in ethanol, 5 M) was continuously fed into the reaction mixture containing ethanol as cosolvent (20%, v/v) to ensure an optimal reaction rate by adjusting the feeding rate and simultaneously increasing the enantioselectivity due to the increased concentration of ethanol. Finally, a maximum of 415 mM of product was produced with an enantiomeric excess value of 97.6% ee. The hydrolysis process was easily scaled up to 2 L, demonstrating that the described biocatalytic process was rationally designed and could be applied further on an industrial scale.
Co-reporter:Erzheng Su and Dongzhi Wei
Journal of Agricultural and Food Chemistry 2014 Volume 62(Issue 27) pp:6375-6381
Publication Date(Web):June 23, 2014
DOI:10.1021/jf501993v
In this work, the low cost naturally immobilized Carica papaya lipase (CPL) was investigated for production of fatty acid butyl esters (FABE) to fulfill the aim of reducing the lipase cost in the enzymatic butyl-biodiesel process. The CPL showed specificities to different alcohol acyl acceptors. Alcohols with more than three carbon atoms did not have negative effects on the CPL activity. The CPL catalyzed butanolysis for FABE production was systematically investigated. The reaction solvent, alcohol/oil molar ratio, enzyme amount, reaction temperature, and water activity all affected the butanolysis process. Under the optimized conditions, the highest conversion of 96% could be attained in 24 h. These optimal conditions were further applied to CPL catalyzed butanolysis of other vegetable oils. All of them showed very high conversion. The CPL packed-bed reactor was further developed, and could be operated continuously for more than 150 h. All of these results showed that the low cost Carica papaya lipase can be used as a promising lipase for biodiesel production.
Co-reporter:Nan Liu, Xiangzhao Mao, Zongjun Du, Bozhong Mu, Dongzhi Wei
Carbohydrate Research 2014 Volume 388() pp:147-151
Publication Date(Web):31 March 2014
DOI:10.1016/j.carres.2014.02.019
•We cloned and characterised a novel β-agarase from Agarivorans gilvus WH0801.•Compared with other known β-agarases, the identity of amino acid sequence was 53%.•The agarase was purified and characterised.•An exo-glycoside agarase which produced neoagarotetraose was first revealed.•This study laid a foundation for the preparation of neoagarotetraose in future.AgWH50A, a novel β-agarase, was cloned from Agarivorans gilvus WH0801 by degenerate and nested PCR. It consists of 942 amino acids (105 kDa), including a 21-amino acid signal peptide. AgWH50A shares the highest amino acid sequence homology with AgaD02 from Agarivorans sp. QM38 (53%). The recombinant agarase gene was expressed in Escherichia coli and purified by affinity chromatography. Maximum enzymatic activity (Km 5.97 mg/mL and Vmax 0.781 U/mg) was observed at pH 6.0 and 30 °C. Using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography, we analysed the hydrolysis products and concluded that AgWH50A is a neoagarotetraose-forming β-agarase, which can cleave agarose into neoagarotetraose. This novel agarase has potential applications in the industrial production of neoagarotetraose and provides a new agarose hydrolysis model for future research.
Co-reporter:Jian Qiu, Erzheng Su, Wei Wang, Dongzhi Wei
Tetrahedron Letters 2014 Volume 55(Issue 8) pp:1448-1451
Publication Date(Web):19 February 2014
DOI:10.1016/j.tetlet.2014.01.044
A strategy of nitrilase mediated dynamic kinetic resolution toward the synthesis of d-phenylglycine was developed, using aqueous-1-octanol biphasic system. Due to the efficient suppression of the decomposition of phenylglycinonitrile, a maximum yield of 81% is obtained. This result indicates that the nitrilase mediated dynamic kinetic resolution is a promising method toward the synthesis of d-phenylglycine and its derivatives.
Co-reporter:Xu Liu;Rong Chen;Zhongwei Yang;Jiale Wang;Jinping Lin
Molecular Biotechnology 2014 Volume 56( Issue 4) pp:285-295
Publication Date(Web):2014 April
DOI:10.1007/s12033-013-9707-z
A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD+/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of α-diketones, hydroxy ketones, α-ketoesters, and β-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various β-ketones and β-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH).
Co-reporter:Wei Wei;Shu-Yue Fan;Feng-Qing Wang
World Journal of Microbiology and Biotechnology 2014 Volume 30( Issue 7) pp:1947-1954
Publication Date(Web):2014 July
DOI:10.1007/s11274-014-1614-3
Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ1-dehydrogenase genes (kstDM and kstDA) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstDM and kstDA were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstDM/kstDA overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60 %, respectively. This study on the manipulation of the main kstDM gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity.
Co-reporter:Jian Qiu;Er-Zheng Su;Hua-Lei Wang;Wen-Wen Cai
Applied Biochemistry and Biotechnology 2014 Volume 173( Issue 2) pp:365-377
Publication Date(Web):2014 May
DOI:10.1007/s12010-014-0845-y
In this study, a high (R)-enantioselective nitrilase gene from Sphingomonas wittichii RW1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The recombinant nitrilase was purified to homogeneity with a molecular weight of 40 kDa. The pH and temperature optima were shown to be pH 8.0 and 40 °C, respectively. The purified nitrilase was most active toward succinonitrile, approximately 30-fold higher than that for phenylglycinonitrile. Using the E. coli BL21/ReSWRW1 whole cells as biocatalysts, the kinetic resolution for asymmetric synthesis of (R)-phenylglycine was investigated at pH 6.0. A yield of 46 % was obtained with 95 % enantiomeric excess (ee), which made it a promising biocatalyst for synthesis of (R)-phenylglycine.
Co-reporter:Wei Wang;Fanju Meng;Pei Liu;Shengli Yang
Journal of Industrial Microbiology & Biotechnology 2014 Volume 41( Issue 11) pp:1709-1718
Publication Date(Web):2014 November
DOI:10.1007/s10295-014-1508-2
Trichoderma reesei is the preferred organism for producing industrial cellulases. However, cellulases derived from T. reesei have their highest activity at acidic pH. When the pH value increased above 7, the enzyme activities almost disappeared, thereby limiting the application of fungal cellulases under neutral or alkaline conditions. A lot of heterologous alkaline cellulases have been successfully expressed in T. reesei to improve its cellulolytic profile. To our knowledge, there are few reports describing the co-expression of two or more heterologous cellulases in T. reesei. We designed and constructed a promoter collection for gene expression and co-expression in T. reesei. Taking alkaline cellulase as a reporter gene, we assessed our promoters with strengths ranging from 4 to 106 % as compared to the pWEF31 expression vector (Lv D, Wang W, Wei D (2012) Construction of two vectors for gene expression in Trichoderma reesei. Plasmid 67(1):67–71). The promoter collection was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. We observed higher activities of both cellulose degradation and biostoning by the co-expression of an endoglucanase and a cellobiohydrolase than the activities obtained by the expression of only endoglucanase or cellobiohydrolase. This study makes the process of engineering expression of multiple genes easier in T. reesei.
Co-reporter:Chaogang Bai;Xiaojuan Wang;Jian Zhang;Aiyou Sun
Biotechnology Letters 2014 Volume 36( Issue 8) pp:1711-1716
Publication Date(Web):2014 August
DOI:10.1007/s10529-014-1535-0
Interleukin-24 (IL-24) is a novel cytokine selectively inhibiting proliferation of cancer cells but with little effect on normal cells. However, IL-24 is difficult to express in Escherichia coli. In this study, we optimised the secondary structure of the translation initiation region using computational approach to obtain non-fusion recombinant IL-24 (nrIL-24). The Gibbs free energy of the region was decreased from −22 to −9.07 kcal mol−1, potentially promoting a loose secondary structure formation and improving the translation initiation efficiency. As a result, the expression of nrIL-24 was increased to 26 % of the total cellular protein from being barely initially detectable. nrIL-24 showed a concentration-dependent inhibition of A375 cells but had little effect on normal human cells. These results demonstrate that this method in increasing nrIL-24 expression is effective and efficient.
Co-reporter:Rui Xu;Yuguo Dong;Lihua Wang;Xinyi Tao
Applied Microbiology and Biotechnology 2014 Volume 98( Issue 23) pp:9633-9641
Publication Date(Web):2014 December
DOI:10.1007/s00253-014-6021-z
Rho GDP dissociation inhibitor 2 (RhoGDI2) was identified as a functional metastasis suppressor in human bladder cancer, suggesting that increasing the RhoGDI2 level may represent a promising therapeutic strategy. It has been shown that the transactivator of transcription (TAT) protein from HIV-1 is able to efficiently deliver various biological molecules into several cell types. In this study, TAT peptide was fused with the N-terminus of RhoGDI2, and the resulting TAT-RhoGDI2 fragment was inserted into the pGEX-6p-1 plasmid and expressed as a glutathione S-transferase (GST)/TAT-RhoGDI2 fusion protein in Escherichia coli BL21(DE3) cells. A two-step purification strategy involving glutathione sepharose chromatography and PreScission protease cleavage was developed to purify TAT-RhoGDI2; subsequently, the identification of the involved macromolecules was achieved by Western blot. The final product, TAT-RhoGDI2, was obtained at a concentration of 112 mg/L. This is the first report on the efficient production of bioactive TAT-RhoGDI2 through a gene-engineering approach in E. coli. Using flow cytometry, we found that the TAT-RhoGDI2 fusion proteins could penetrate into bladder cancer cells with an extremely high efficiency. In vitro scratch and transwell assay and the migration/invasion behavior of UMUC3 cells were strongly reduced by the treatment with TAT-RhoGDI2. These studies support the use of the TAT-RhoGDI2 protein in tumor metastasis therapy.
Co-reporter:Senwen Deng;Erzheng Su;Xiaoqiang Ma
Bioprocess and Biosystems Engineering 2014 Volume 37( Issue 8) pp:1517-1526
Publication Date(Web):2014 August
DOI:10.1007/s00449-013-1123-z
d-Amino acid oxidase is an important biocatalyst used in a variety of fields, and its economically justified level recombinant expression in Escherichia coli has not been established. To accomplish this, after a single Phe54Tyr substitution, fusion proteins of d-amino acid oxidase from Trigonopsis variabilis (TvDAO) with 6 × His-tags were constructed and expressed in E. coli. The effects of his-tags fusing position were revealed. Significant increase in holoenzyme percent and protein solubility made N-terminus tagged TvDAO (termed NHDAO) a suitable choice for TvDAO production. However, reduced cell growth and protein production rates were also observed for the NHDAO bearing strains. To optimize the performance of NHDAO production, changes of culture medium were tested. Finally, a production of 140 U/mL or 3.48 g active enzyme per liter which accounted for 41.4 % of the total protein, and a specific activity of 16.68 U/mg for the crude extract, were achieved in a 3.7 L fermenter in 28.5 h. This indicated a possibility for functional and economical TvDAO expression in E. coli to meet the industrial need.
Co-reporter:Dr. Xin Gao;Shuai Yang;Chengcheng Zhao; Yuhong Ren; Dongzhi Wei
Angewandte Chemie International Edition 2014 Volume 53( Issue 51) pp:14027-14030
Publication Date(Web):
DOI:10.1002/anie.201405016
Abstract
A strategy for scaffold-free self-assembly of multiple oligomeric enzymes was developed by exploiting enzyme oligomerization and protein–protein interaction properties, and was tested both in vitro and in vivo. Octameric leucine dehydrogenase and dimeric formate dehydrogenase were fused to a PDZ (PSD95/Dlg1/zo-1) domain and its ligand, respectively. The fusion proteins self-assembled into extended supramolecular interaction networks. Scanning-electron and atomic-force microscopy showed that the assemblies assumed two-dimensional layer-like structures. A fluorescence complementation assay indicated that the assemblies were localized to the poles of cells. Moreover, both in vitro and in vivo assemblies showed higher NAD(H) recycling efficiency and structural stability than did unassembled structures when applied to a coenzyme recycling system. This work provides a novel method for developing artificial multienzyme supramolecular devices and for compartmentalizing metabolic enzyme cascades in living cells.
Co-reporter:Xi-Hua Zhao;Dong-Zhi Wei;Wei Wang
Applied Biochemistry and Biotechnology 2014 Volume 173( Issue 6) pp:1295-1302
Publication Date(Web):2014 July
DOI:10.1007/s12010-014-0924-0
Here we cloned and expressed two alkaline β-1, 4-endoglucanases of Phaeosphaeria sp. LH21 from deep-sea mud. The two enzymes shared 71 and 63 % of identities with their known β-1, 4-endoglucanases, respectively. According to the primary and spatial structures, the potential active sites of one of the two enzymes could be Asp122 and Asp11, while the other enzyme could be Asp16. The enzymatic properties of their recombinant enzymes from Pichia pastoris GS115 showed that they were optimally active at pH 8 and 60–65 °C, exhibited >90 % residual relative activities at pH 3–10, and obtained relative activities >75 % at pH 5–10.
Co-reporter:Nan Liu;Xiangzhao Mao;Meng Yang
World Journal of Microbiology and Biotechnology 2014 Volume 30( Issue 6) pp:1691-1698
Publication Date(Web):2014 June
DOI:10.1007/s11274-013-1591-y
agWH50C, a novel β-agarase gene, was cloned from Agarivorans gilvus WH0801 by degenerate PCR and nested PCR. The gene agWH50C comprized a 2,223-bp, encoding a protein of 740 amino acids. Sequencing results demonstrated that AgWH50C shared 45 % sequence identity with a well characterized β-agarase, Aga50D, from Saccharophagus degradans 2–40. The mature agarase was expressed in Escherichia coli and purified by affinity chromatography. The optimum pH and temperature for AgWH50C activity were 6.0 and 30 °C. The Km and Vmax values for agarose were 12.55 mg/ml and 1.17 U/mg. Analysis of the hydrolysis products using linear ion trap mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography confirmed that the reaction product of AgWH50c was α-neoagarobiose alone. Therefore, our novel agarase has the potential for industrial applications to produce neoagarobiose as well as provides a key β-agarase for fermentation of agar biomass.
Co-reporter:Lujia Zhang, Bei Gao, Zuanning Yuan, Xiao He, Y. Adam Yuan, John Z.H. Zhang, Dongzhi Wei
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2014 Volume 1844(Issue 7) pp:1183-1192
Publication Date(Web):July 2014
DOI:10.1016/j.bbapap.2014.02.021
•The structure and catalytic mechanism of a novel family I.1 lipase was determined.•A simple strategy for manipulating the chiral selectivity was improved.•A crucial distance in enzyme-substrate complex to judge virtual mutations was identified.•A novel electrostatic interaction analysis protocol was used to design mutants.•All the computational designs matched perfectly with the experimental results.Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme–substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.
Co-reporter:Daihui Zhang, Chong Wang, Hongxi Wu, Jingli Xie, Lei Du, Zhaoming Xia, Jingbo Cai, Zhenhua Huang, Dongzhi Wei
Food Chemistry 2013 Volume 138(Issue 1) pp:306-314
Publication Date(Web):1 May 2013
DOI:10.1016/j.foodchem.2012.10.061
Three sulphated polysaccharides, coded as BEMPA, BEMPB1, BEMPB2, were extracted from the mucilage of mud snail of Bullacta exarata and purified by DEAE-cellulose ion-exchange and size-exclusion chromatography. Structural analysis of purified polysaccharides by chemical and biochemical methods revealed BEMPA was a high (1→3,4)-linked mannose-containing polysaccharide with molecular weight of 22,977 Da. BEMPB1, with molecular weight of 64,117 Da, was a high (1→3)-linked arabinose-containing polysaccharide. BEMPB2 was mainly composed of (1→3,4)-linked mannose with molecular weight of 47,507 Da. The comparison between sulphated polysaccharides and their desulphated products showed that sulphate substitutions of BEMPB1 were deduced to be at the C-3 of (1→4)-linked mannose, while sulphate substitutions of BEMPA and BEMPB2 were at C-4 of (1→3)-linked mannose. Furthermore, BEMPA exhibited highest inhibitory effects on growth of B-16 melanoma cells, and IC50 were 31.1 μg/mL.Highlights► Polysaccharides from the mucilage of mud snail were studied for the first time. ► They were sulphated polysaccharides. ► Chemical composition and structure feature were remarkably distinct. ► BEMPA showed significant inhibitory effects on the growth of cancer cells.
Co-reporter:Pengyong You;Jian Qiu;Erzheng Su
European Journal of Organic Chemistry 2013 Volume 2013( Issue 3) pp:557-565
Publication Date(Web):
DOI:10.1002/ejoc.201201055
Abstract
An efficient synthesis of the (S)-3-amino-3-phenylpropanoic acid enantiomer has been achieved by Carica papaya lipase (CPL) catalysed enantioselective alcoholysis of the corresponding racemic N-protected 2,2,2-trifluoroethyl esters in an organic solvent. A high enantioselectivity (E > 200) was achieved by two strategies that involved engineering of the substrates and optimization of the reaction conditions. Based on the resolution of a series of amino acids, it was found that the structure of the substrate has a profound effect on the CPL-catalysed resolution. The enantioselectivity and reaction rate were significantly enhanced by switching the conventional methyl ester to an activated trifluoroethyl ester. When considering steric effects, the substituted phenyl and amino groups should not both be large for the CPL-catalysed resolution. The mechanism of the CPL-catalysed enantioselective alcoholoysis of the amino acids is discussed to delineate the substrate requirements for CPL-catalysed resolution. Finally, the reaction was scaled up, and the products were separated and obtained in good yields (≥ 80 %). The (S)-3-amino-3-phenylpropanoic acid obtained was used as a key chiral intermediate in the synthesis of (S)-dapoxetine with very high enantiomeric excess (> 99 %).
Co-reporter:Xi-Hua Zhao;Wei Wang;Dong-Zhi Wei
Journal of Microbiology 2013 Volume 51( Issue 1) pp:82-87
Publication Date(Web):2013 February
DOI:10.1007/s12275-013-2370-z
The phylogenetic tree of the partial elongation factor-1 alpha gene fits better than the partial 18S rDNA for generic classification. From the results of the molecular tree and analysis of morphological characters, Petriella setifera LH was identified. It can be induced to produce carboxymethyl cellulase (CMCase). The crude CMCase only shows a 44.1-kDa band by activity staining after SDS-PAGE. It is optimally active at 55°C and pH 6.0, and is stable from pH 5.0–8.0 and at 45°C or below. The crude CMCase, which is not affected by Co2+, is strongly activated in the presence of 10 mM Na+, K+, Ca2+, Mg2+, EDTA, and Mn2+. It is strongly inhibited by 10 mM Fe2+, Pb2+, Al3+, Zn2+, Ag+, Fe3+, and Cu2+. When compared with denim treatment by Novoprime A800 (a commercial neutral cellulase), crude CMCase exhibits a similar fabric weight loss and indigo dye removal. These results indicate that crude CMCase has potential application in denim biostoning.
Co-reporter:Jianan Sun;Liaoyuan Zhang;Ben Rao;Yunbin Han
Biotechnology and Bioprocess Engineering 2012 Volume 17( Issue 3) pp:598-605
Publication Date(Web):2012 June
DOI:10.1007/s12257-011-0587-4
Enhanced acetoin production was carried out by Serratia marcescens H32. First, medium compositions were optimized statistically for shake flask fermentations to produce acetoin. Sucrose and corn steep liquor powder (CSLP) were identified as the most significant factors by Plackett-Burman design. The path of steepest ascent and response surface methodology were then employed to determine the optimal concentrations of the two factors. Acetoin yield was up to 41.5 g/L in flask fermentations using the optimized medium. Furthermore, the optimal medium was used to conduct fermentation experiments in a 3.7-L bioreactor. The influences of different agitation speeds on acetoin production were investigated. Based on a process analysis, a two-stage agitation speed control strategy was proposed, in which the agitation speed was controlled at 700 rpm during the first 8 h and then switched to 600 rpm. A relatively high acetoin concentration (44.9 g/L) and high acetoin productivity (1.73 g/L/h) were achieved by applying this strategy. Fed-batch fermentation based on the two-stage agitation speed control strategy was performed, and a maximum acetoin concentration of 60.5 g/L with productivity of 1.44 g/L/h was achieved.
Co-reporter:Liu-Jing Wei;Ji-lai Zhou;Dan-ni Zhu
Biotechnology and Bioprocess Engineering 2012 Volume 17( Issue 6) pp:1156-1164
Publication Date(Web):2012 December
DOI:10.1007/s12257-012-0339-0
In this study a new insight was provided to understand the functions of membrane-bound alcohol dehydrogenase (mADH) and aldehyde dehydrogenase (mALDH) in the bio-oxidation of primary alcohols, diols and poly alcohols using the resting cells of Gluconobacter oxydans DSM 2003 and its mutant strains as catalyst. The results demonstrated that though both mADH and mALDH participated in most of the oxidation of alcohols to their corresponding acid, the exact roles of these enzymes in each reaction might be different. For example, mADH played a key role in the oxidation of diols to its corresponding organic acid in G. oxydans, but it was dispensable when the primary alcohols were used as substrates. In contrast to mADH, mALDH appears to play a relatively minor role in organic acid-producing reactions because of the possible presence of other isoenzymes. Aldehydes were, however, found to be accumulated in the mALDH-deficient strain during the oxidation of alcohols.
Co-reporter:Miao-Miao Chen;Feng-Qing Wang;Liang-Cai Lin
Applied Microbiology and Biotechnology 2012 Volume 96( Issue 1) pp:133-142
Publication Date(Web):2012 October
DOI:10.1007/s00253-011-3855-5
Microbial ∆1-dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid-∆1-dehydrogenase (kstDF) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstDF encodes a polypeptide consisting of 637 amino acid residues. It shows 51% amino acid identity with a kstD from Thermomicrobium roseum DSM 5159. Expression of kstDF in Escherichia coli and Pichia pastoris showed that all kstDF activity is located in the cytoplasm. This indicates that it is a soluble intracytoplasmic enzyme, unlike most kstDs from bacteria, which are membrane-bound. The expression of kstDF was performed in P. pastoris, both intracellularly and extracelluarly. The intracellularly expressed protein displayed good activity in steroid transformation, while the extracellularly expressed protein showed nothing. Interestingly, the engineered P. pastoris KM71 (KM71I) and GS115 (GS115I) showed different transformation activities for 4-androstene-3,17-dione (AD) when kstDF was expressed intracellularly. Under the same conditions, KM71I was found capable of transforming 1.0 g/l AD to 1,4-androstadiene-3,17-dione (ADD), while GS115I could transform 1.5 g/l AD to both ADD and boldenone (BD). The production of BD is attributed to a 17β-hydroxysteroid dehydrogenase in P. pastoris GS115I, which catalyzes the reversible reaction between C17-one and C17-alcohol of steroids. The conversion of AD by GS115I and KM71I may provide alternative means of preparing ADD or BD. In brief, we show here that kstDF is a promising enzyme in steroid ∆1-dehydrogenation that is propitious to construct genetically engineered steroid-transforming recombinants by heterologous overexpression.
Co-reporter:Xiangyu Gong, Erzheng Su, Pixiang Wang, Dongzhi Wei
Tetrahedron Letters 2011 Volume 52(Issue 41) pp:5398-5402
Publication Date(Web):12 October 2011
DOI:10.1016/j.tetlet.2011.08.056
A new strategy based on enantioselective acylation properties of relatively unknown penicillin G acylase from Alcaligenes faecalis has been developed for the production of pharmacologically interesting enantiomerically pure d-phenylalanine. In order to get high reaction rate and enantioselectivity, two key factors (pH and temperature) and eight different acyl donors were optimized, and the optimal acylation reaction was carried out at pH 10, 35 °C, using phenylacetamide as the acyl donor. This enantioselective acylating method is also illustrated by the effective production of five different p-substituted phenylalanine derivatives in enantiopure.
Co-reporter:Jian Wu;Ming Hua Li;Jin Ping Lin;Dong Zhi Wei
Current Microbiology 2011 Volume 62( Issue 4) pp:1123-1127
Publication Date(Web):2011 April
DOI:10.1007/s00284-010-9831-y
The Gluconobacter oxydans M5 with disruption of the pyrroloquinoline quinine-dependent membrane-bound aldehyde dehydrogenase (ALDH) was used for the oxidation of benzyl alcohol. The selectivity toward benzaldehyde showed an obvious increase for the engineered strain, which reached the 67.3%, while the wild strain had only 2.8%. Meantime, the aqueous/isooctane (1:1) biphasic system was used for the further improvement of selectivity. By these methods, nearly 100% selectivity and conversion rate could be obtained within 1 h at the optimum initial benzyl alcohol concentration of 5.0 g/l.
Co-reporter:Yanlong Xing;Shu Wang;Xiangzhao Mao;Xuebo Zhao
Journal of Fluorescence 2011 Volume 21( Issue 2) pp:587-594
Publication Date(Web):2011 March
DOI:10.1007/s10895-010-0746-6
Water-soluble aldehydes (acetaldehyde, propionaldehyde) and non-water-soluble aldehydes (butyraldehyde and phenylacetaldehyde) were easily detected by an efficient fluorescent method with 5-aminofluorescein as probe. Under optimal detection conditions, 5-aminofluorescein could selectively respond to aldehydes with high sensitivity in comparison with other carbonyl compounds like ketones and acids. Thus, the proposed method was used to monitor microbial oxidation and succeeded in trapping transiently-produced aldehydes during biotransformation of primary alcohols by Gluconobacter oxydans.
Co-reporter:Xiangzhao Mao;Shi Chen;Yaling Shen
World Journal of Microbiology and Biotechnology 2011 Volume 27( Issue 9) pp:2033-2039
Publication Date(Web):2011 September
DOI:10.1007/s11274-011-0665-y
Recently the biosynthetic mechanism of FR-008/Candicidin was gradually revealed, and a whole gene cluster responsible for its biosynthesis in Streptomyces sp. FR-008 was cloned and identified. A series of novel FR-008/Candicidin derivatives were obtained by a combination of biochemical methods, which have potential application in medicine and agriculture. The yield of the antibiotic was enhanced twofold when adding CuSO4 to the culture medium at a concentration of 42.8 mg/l in a 3.7 l fermentor, which led to more rapid glucose consumption and decreased cell growth. Enzyme activities related to glucose metabolism were enhanced greatly by copper addition. Transcriptional analysis of samples in the fermentation process demonstrated adding copper sulfate to the fermentation medium could bring a large increase of transcriptional level of the FR-008/Candicidin biosynthetic genes fscA, fscB, fscC and fscD. This study demonstrated that the copper ion played a crucial role in antibiotic biosynthesis and was understood to be related to the key enzyme activities and the gene transcriptional levels. These results would be very useful for maximizing productivity of the antibiotic in an upscaled fermentation.
Co-reporter:Ren Peng;Jinping Lin
Applied Biochemistry and Biotechnology 2011 Volume 165( Issue 3-4) pp:926-937
Publication Date(Web):2011 October
DOI:10.1007/s12010-011-9309-9
The genes of CS-2 lipase and its cognate foldase were cloned from Pseudomonas aeruginosa CS-2. A stop codon was not found in the lipase gene. The amino acid sequence deduced from the lipase gene from P. aeruginosa CS-2 showed 97.8%, 71.3%, and 71.2% identity with lipases from P. aeruginosa LST-03, Pseudomonas mendocina ymp, and Pseudomonas stutzeri A1501, respectively. The co-expression of CS-2 lipase and its cognate foldase of P. aeruginosa CS-2 in Escherichia coli BL21 (DE3) resulted in the formation of a soluble lipase. The recombinant lipase and foldase were purified to homogeneity using nickel affinity chromatography and about 10.2-fold with 40.9% recovery was achieved for the purification of the recombinant lipase. The molecular masses of the lipase and the foldase were estimated to be 35.7 and 38.3 kDa in SDS-PAGE, respectively. The recombinant lipase showed stability in the presence of some organic solvents. The recombinant CS-2 lipase was immobilized and subsequently used for the synthesis of butyl acetate in heptane. The conversion of substrate decreased from 98.2% to 87.4% after 5 cycles in reuse of the immobilized lipase.
Co-reporter:Shu Wang;Xiangzhao Mao;Hualei Wang;Jinping Lin
Applied Microbiology and Biotechnology 2011 Volume 91( Issue 2) pp:287-294
Publication Date(Web):2011 July
DOI:10.1007/s00253-011-3267-6
A novel water-soluble dextran was synthesized from maltodextrin by cell-free extract of Gluconobacter oxydans DSM 2003. The dextran was purified by size exclusion chromatography, and the structure was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and gas chromatography–mass spectrometer. Based on the spectral data, we found that the dextran contained only d-glucose residues. The ratio of nonreducing end glucopyranosyl (Glcp) to 6-linked Glcp to 4,6-linked Glcp was estimated to be 8.62:78.79:12.59 by methylation analysis. This result indicated the existence of a small proportion of α(1,4) branches in α(1,6) glucosyl linear chains. Here, we reported the first time a novel dextran was synthesized by G. oxydans DSM 2003.
Co-reporter:Er-zheng Su 苏二正;Ying Zhou 周 英
Journal of Shanghai University (English Edition) 2010 Volume 14( Issue 2) pp:137-144
Publication Date(Web):2010 April
DOI:10.1007/s11741-010-0212-2
The lipase activities in the three main Chinese castor bean varieties [ZiBi 05 (ZB 5), TongBi 06 (TB 6), JinBi 02 (JB 2)] were investigated. Two lipases were found in extracts from the endosperms of all the three varieties. One lipase is located in the lipid bodies, showing higher activity at pH 4.5 and exhibiting the highest activity at day 1 of germination. The other lipase is located in the particulates, showing higher activity at pH 9.5 and particularly active at day 4 of germination. The lipid bodies from ZB 5 show the highest lipase activity at day 1 and pH 4.5 among the three varieties during the 8 d of germination. The acid lipase located in the lipid bodies of ZB 5 of germination 1 d was purified to homogeneity by DEAE-sepharose CL-6B and butyl-sepharose CL-4B chromatography after isolation, delipidation and solubilization, with a purification factor of 16.1 and overall activity recovery of 17.7%. The characterizations (such as optimum pH, pH stability, optimum temperature, thermal stability, effect of metal ions, organic solvents stability, substrate specificity) of the purified acid lipase were conducted in detail. A combination of the characterized properties may make this enzyme a useful biocatalyst for industrial application.
Co-reporter:Minghua Li;Jian Wu;Jinping Lin
Current Microbiology 2010 Volume 61( Issue 5) pp:370-375
Publication Date(Web):2010 November
DOI:10.1007/s00284-010-9621-6
Dihydroxyacetone (DHA) is an important ketose sugar, which is extensively used in the cosmetic, chemical, and pharmaceutical industries. DHA has been industrially produced by Gluconobacter oxydans with a high demand of oxygen. To improve the production of DHA, the gene vgb encoding Vitreoscilla hemoglobin (VHb) was successfully introduced into G. oxydans, where it was stably maintained, and expressed at about 76.0 nmol/g dry cell weight. Results indicated that the constitutively expressed VHb improved cell growth and DHA production in G. oxydans under different aeration conditions. Especially at low aeration rates, the VHb-expressing strain (VHb+) displayed 23.13% more biomass and 37.36% more DHA production than those of VHb-free strain (VHb−) after 32 h fermentation in bioreactors. In addition, oxygen uptake rate (OUR) was also increased in VHb+ strain relative to the control strain during fermentation processes.
Co-reporter:Liujing Wei;Xuepeng Yang;Keliang Gao;Jinping Lin
Molecular Biotechnology 2010 Volume 46( Issue 1) pp:26-33
Publication Date(Web):2010 September
DOI:10.1007/s12033-010-9263-8
Although Gluconobacter oxydans can convert 1,2-propanediol to d-(−)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of d-(−)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in d-(−)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to d-(−)-lactic acid by G. oxydans DSM 2003.
Co-reporter:Bo Li;Wei Wang;Feng-Qing Wang;Dong-Zhi Wei
Applied Microbiology and Biotechnology 2010 Volume 85( Issue 6) pp:1831-1838
Publication Date(Web):2010 February
DOI:10.1007/s00253-009-2188-0
Diosgenin transformation was studied in Streptomyces virginiae IBL-14, a soil-dwelling bacterium with diosgenin-degrading capacity. All of the derivatives isolated were identified as 4-ene-3-keto steroids. We cloned ChoL, a fragment of a cholesterol oxidase from S. virginiae IBL-14, and used gene-disruption techniques to determine its function in the oxidation of diosgenin to 4-ene-3-keto steroids. Subsequently, the entire open reading frame of ChoL was cloned by chromosome walking, and the His6-tagged recombinant protein was overproduced, purified, and characterized. ChoL consisted of 1,629 nucleotides that encoded a protein of 542 amino acids, including a 34-residue putative signal peptide at the N-terminal. ChoL showed 85% amino acid similarity to ChoA from Streptomyces sp. SA-COO. This enzyme can also oxidize other steroids such as cholesterol, sitosterol, and dehydroepiandrosterone, which showed higher affinity (Km = 0.195 mM) to diosgenin. The catalytic properties of this enzyme indicate that it may be useful in diosgenin transformation, degradation, and assay.
Co-reporter:Liaoyuan Zhang;Jian’an Sun;Yingli Hao
Journal of Industrial Microbiology & Biotechnology 2010 Volume 37( Issue 8) pp:857-862
Publication Date(Web):2010 August
DOI:10.1007/s10295-010-0733-6
Serrawettin W1 produced by Serratia marcescens is a surface-active exolipid resulting in a lot foam formation during the 2,3-butanediol (2,3-BD) fermentation process. In order to avoid excessive addition of antifoam agent and microbial contamination, S. marcescens mutants deficient in serrawettin W1 formation were successfully constructed through insertional inactivation of the swrW gene coding for serrawettin W1 synthase. The shake flask and batch experiments suggested that disruption of the swrW gene led to significant reduction of the foam formation and improved 2,3-BD production a little. Ultimately, fed-batch culturing of the mutant afforded a maximum 2,3-BD concentration of 152 g l−1 with a productivity of 2.67 g l−1 h−1 and a yield of 92.6% at 57 h.
Co-reporter:Xue-Peng Yang;Gui-Fang Zhong;Jin-Ping Lin
Journal of Industrial Microbiology & Biotechnology 2010 Volume 37( Issue 6) pp:575-580
Publication Date(Web):2010 June
DOI:10.1007/s10295-010-0703-z
We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydanstldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation—up to 6 mM—in the medium. This yield is 21.5-fold higher than that obtained in previous studies.
Co-reporter:Qiang Tan;Yewang Zhang;Qingxun Song
World Journal of Microbiology and Biotechnology 2010 Volume 26( Issue 1) pp:
Publication Date(Web):2010 January
DOI:10.1007/s11274-009-0153-9
In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min−1, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h−1 to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.
Co-reporter:Li Zhao;Xingyuan Ma;Yu Zheng;Jianguo Zhang;Guodong Wei
Journal of Chemical Technology and Biotechnology 2009 Volume 84( Issue 4) pp:626-632
Publication Date(Web):
DOI:10.1002/jctb.2092
Abstract
BACKGROUND: Glycerol dehydrogenase [EC.1.1.1.6] and 1,3-propanediol oxidoreductase [EC.1.1.1.202] were proved to be two of the key enzymes for glycerol conversion to 1,3-propanediol in Klebsiella pneumoniae under anaerobic conditions. For insight into their significance on 1,3-propanediol production under micro-aerobic conditions, these two enzymes were over-expressed in K. pneumoniae individually, and their effects on conversion of glycerol into 1,3-propanediol in a resting cell system under micro-aerobic conditions were investigated.
RESULTS: In the resting cell system, over-expression of 1,3-propanediol oxidoreductase led to faster glycerol conversion and 1,3-propanediol production. After a 12 h conversion process, it improved the yield of 1,3-propanediol by 20.4% (222.1 mmol L−1 versus 184.4 mmol L−1) and enhanced the conversion ratio of glycerol into 1,3-propanediol from 50.8% to 59.8% (mol mol−1). Over-expression of glycerol dehydrogenase in K. pneumoniae had no significant influence both on 1,3-propanediol yield and on the conversion ratio of glycerol into 1,3-propanediol in the resting cell system.
CONCLUSION: The results were important for an understanding of the significance of glycerol dehydrogenase and 1,3-propanediol oxidoreductase in 1,3-proanediol production under micro-aerobic conditions, and for developing better strategies to improve 1,3-propanediol yield. Copyright © 2008 Society of Chemical Industry
Co-reporter:Jun-hua Shi;Jun-hua Xiao;Dong-zhi Wei
Medicinal Chemistry Research 2009 Volume 18( Issue 7) pp:538-544
Publication Date(Web):2009 September
DOI:10.1007/s00044-008-9147-7
18β-Glycyrrhetinic acid (GA) is the active component in licorice root. Recent study has shown that GA exhibits many pharmacological activities. Here a new derivative of GA, biotinylated GA (BGA), was synthesized by introducing the biotin to the C-29 carboxyl of GA, and its antitumor effects were confirmed through tests on mouse B16 melanoma cells and BEL 7402 human hepatocarcinoma cells. At the same time, it is shown that the biotin group in BGA has no influence on the antitumor effects of GA. Then the stability of BGA in Roswell Park Memorial Institute (RPMI) medium 1640 and water was determined by high-performance liquid chromatography (HPLC), demonstrating much greater stability than GA in these two liquids.
Co-reporter:Hengwei Wang;Fengqing Wang
Applied Microbiology and Biotechnology 2009 Volume 82( Issue 2) pp:249-259
Publication Date(Web):2009 February
DOI:10.1007/s00253-008-1756-z
In shake flasks, good oxygen supply tended to decrease rtPA expression in media containing only yeast extract and tryptone, while oxygen limitation would increase rtPA synthesis in the same medium. Our data showed that though the drop of rtPA expression in the 20-ml cultures of LBG or 2YTG was accompanied with a severe acetate accumulation, it was actually caused by low ammonia. The rtPA expression level could be significantly improved by increasing culture ammonium ion up to 500 mM. The effects of exogenous high ammonia on cell growth and rtPA expression were further examined in shake flasks and a 4-l fermentor. The high ammonia had no significant impact on cell growth and oxygen respiratory activity but significantly depressed the activities of glutamine synthetase/glutamate synthase and glutamate dehydrogenase, suggesting that ammonium ion as a nitrogen source improved the protein expression by mediating ammonia-assimilating enzymes. We thus propose in our work that E. coli cells, which were grown to a certain density to produce rtPA, would undergo nitrogen starvation under the low ammonia conditions even when the organic nitrogen sources remained abundant. The scale-up of rtPA production from shake flasks to fermentors could be readily achieved in the media containing rich ammonium ion.
Co-reporter:Xiangzhao Mao;Feng Wang;Jianguo Zhang;Shi Chen
Applied Biochemistry and Biotechnology 2009 Volume 159( Issue 3) pp:
Publication Date(Web):2009 December
DOI:10.1007/s12010-008-8502-y
CS103, the novel derivative of polyene macrolides antibiotic FR-008/candicidin with lower toxicity has been isolated from the culture mycelia of the mutant of Streptomyces sp. FR-008, with targeted deletions of the fscP cytochrome P450 gene from its chromosome. To enhance biosynthesis of CS103, pH shift and precursor feeding strategy for fermentation process by the mutant of Streptomyces sp. FR-008 in a stirred tank bioreactor was developed. According to the process parameters analysis, the effectiveness of the strategy was examined and confirmed by experiments. A maximal CS103 concentration of 139.98 μg/mL was obtained, 2.05-fold higher than that in the pH-uncontrolled fermentation. Compared to other three cases as pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch cultures, the proposed “pH shift and precursor feeding strategy” effectively avoided the scarcity of the antibiotic precursor, increased the CS103 yield from biomass (YP/X) and substrate (YP/S) by 110.61% and 48.52%, respectively, and at the time the fermentation time was shortened from 120 to 96 h. The highest CS103 production rate (1.46 μg mL−1 h−1) of the pH shift and precursor feeding strategy was 284.21%, 97.30%, and 58.70% higher than that of pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch culture cases, respectively.
Co-reporter:Li Zhao;Yu Zheng;Xingyuan Ma
Bioprocess and Biosystems Engineering 2009 Volume 32( Issue 3) pp:313-320
Publication Date(Web):2009 April
DOI:10.1007/s00449-008-0250-4
Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.
Co-reporter:Guodong Wei;Xuepeng Yang;Tula Gan
Journal of Industrial Microbiology & Biotechnology 2009 Volume 36( Issue 8) pp:1029-1034
Publication Date(Web):2009 August
DOI:10.1007/s10295-009-0584-1
Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.
Co-reporter:Yu Zheng;Haiyi Zhang;Li Zhao;Liujing Wei;Xingyuan Ma
Journal of Chemical Technology and Biotechnology 2008 Volume 83( Issue 10) pp:1409-1412
Publication Date(Web):
DOI:10.1002/jctb.1958
Abstract
BACKGROUND: 2,3-Butanediol (2,3-BD) is a valuable chemical that can be biosynthesized from many kinds of substrates. For commercial biological production of 2,3-BD, it is desirable to use cheap substrate without pretreatment, such as starch. However, there have been few reports on the production of 2,3-BD directly from starch.
RESULTS: In this work, gene malS coding for α-amylase (EC 3.2.1.1) precursor was inserted into plasmid pUC18K, and secretory over-expression of α-amylase was achieved by engineered Klebsiella pneumoniae. The extracellular recombinant amylase accelerated the hydrolyzation of starch, and one-step production of 2,3-BD from starch was carried out by engineered K. pneumoniae. A 2,3-BD concentration of 3.8 g L−1 and yield of 0.19 g 2,3-BD g−1 starch were obtained after 24 h fermentation.
CONCLUSION: The one-step production of 2,3-BD from starch was achieved by secretory over-expression of amylase in K. pneumoniae. This would simplify the process and reduce the production cost considerably by enabling use of starch with minimal pretreatment. Copyright © 2008 Society of Chemical Industry
Co-reporter:H. Zhu;Y. L. Shen;D. Z. Wei;J. W. Zhu
Current Microbiology 2008 Volume 56( Issue 6) pp:645-650
Publication Date(Web):2008 June
DOI:10.1007/s00284-008-9140-x
Quorum sensing in Serratia marcescens, which uses two types of signaling molecules–N-acyl homoserine lactones and furanosyl borate diester–play important regulatory roles in the synthesis of 2,3-butanediol and prodigiosin. In the hope of understanding the effect of quorum sensing on physiologic metabolism, we established two molecular strategies, one to express acyl-homoserine lactone hydrolase to inactivate AI-1 signaling molecule using an expression vector with lactose as the inducer and the other to mutate luxS gene with a suicide plasmid pUTKm2 to inhibit the synthesis of AI-2 signaling molecule.
Co-reporter:Hu Zhu;Ya-Ling Shen;Dong-Zhi Wei;Jia-Wen Zhu
Molecular and Cellular Biochemistry 2008 Volume 319( Issue 1-2) pp:87-90
Publication Date(Web):2008 December
DOI:10.1007/s11010-008-9880-8
The key genes, SpnI and SpnR, involved in AI-1-quorum sensing system of Serratia marcescens strain H30 were cloned and localized using specific primers (5′-CTTGAACTGTTTGACGTCAGC-3′ and 5′-AGCGGCCAGGTAATAACTGA-3′, 5′-GCCTTCAATGAAAATCAGACC-3′ and 5′-TGTCGCTGTGATAAGCTCCA-3′) designed according to the nucleic acid sequences published at NCBI (accession no. AB234869). The PCR result demonstrated that the genes SpnI and SpnR were located on the bacterial chromosome and plasmid, respectively. This was also confirmed by Southern blotting using an internal fragment (379 bp) from SpnR gene as a probe. These results imply a new type quorum sensing regulation system that had never been reported previously.
Co-reporter:Hu Zhu;Ya-Ling Shen;Dong-Zhi Wei;Jia-Wen Zhu
Molecular and Cellular Biochemistry 2008 Volume 315( Issue 1-2) pp:
Publication Date(Web):2008 August
DOI:10.1007/s11010-008-9784-7
Serratiamarcescens utilizes two types of quorum-sensing signal molecules: N-acyl homoserine lactones and furanosyl borate diester (AI-2). S-adenosylmethionine synthetase (METK), S-adenosylhomocysteine nucleosidase (PFS), and S-ribosylhomocysteinase (LUXS) are three key enzymes in the biosynthetic pathway leading to AI-2 production. The sequence of luxS gene was published at NCBI (Accession number: EF164926). So in this study, Serratia marcescensmetK and pfs genes were successfully cloned with inverse PCR. The results show that the ORF lengths of metK and pfs are 1155 and 702 bp, and encode proteins of 384 and 233 residues. Their molecular weights and isoelectric points are 41.85 kD and 5.50; 27.67 kD and 5.56, which are acidic proteins judging from the calculated pI values. Expression products of two genes with pET28a(+) system exhibited molecular weights in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis comparable with a theoretical estimation. The sequences of these two genes were conferred China patent application numbers CN 200710048016.X and CN 200710048015.5, respectively.
Co-reporter:Jinping Lin;Qinghai Liu;Erzheng Su
Applied Microbiology and Biotechnology 2008 Volume 80( Issue 5) pp:
Publication Date(Web):2008 October
DOI:10.1007/s00253-008-1602-3
Industrial bakers’ yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and d-arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.
Co-reporter:Qiang Tan;Qingxun Song;Yewang Zhang
Applied Biochemistry and Biotechnology 2007 Volume 136( Issue 3) pp:279-289
Publication Date(Web):2007 March
DOI:10.1007/s12010-007-9026-6
The high-density fermentation of recombinant Pichia pastoris was carried out in a 1-L fermentor. After 60 h of fermentation, the activities of d-amino acid oxidase (DAAO) and catalase assayed with the permeabilized cells attained 12,532 and 684,800 U/L, respectively. Additionally, the stability of DAAO and catalase within the permeabilized cells was relatively high. The half-life of the two enzymes reached 14.5 and 4.0 d at 30°C, respectively. Furthermore, these permeabilized cells could convert D-phenylalanine into 99% phenylpyruvate within 100 min and could be efficiently reused up to 13 cycles. After being treated with base and heating, these treated permeabilized cells could be reused up to three cycles in a batchwise conversion of cephalosporin C, and about 90% 7-β-(4-carboxybutanamido)-cephalosporanic acid was ultimately obtained at each cycle.
Co-reporter:Xing Wen Gong, Dong Zhi Wei, Ming Lei He, Yu Chun Xiong
Talanta 2007 Volume 71(Issue 1) pp:381-384
Publication Date(Web):15 January 2007
DOI:10.1016/j.talanta.2006.04.010
Free polyethylene glycol (PEG) is a byproduct produced during the process of pegylation and should be removed for the purification of pegylated proteins. In this paper, it was used to develop a new method for obtaining the modification extent of pegylated proteins. This method included two steps of operation. Firstly, the free PEG was separated from crude reaction mixture of pegylated proteins by CM-Sepharose FF. Then PEG was determined based on the formation of a complex with barium chloride and iodine solution. The effective detective range of PEG was 0–7.5 μg/ml. The modification extent was calculated according to a formula. This method is simple, sensitive, and applicable to all of the PEG derivatives. The most distinctive aspect is that it does not consume proteins.
Co-reporter:Feng-Qing Wang;Bo Li;Wei Wang;Cheng-Gang Zhang
Applied Microbiology and Biotechnology 2007 Volume 77( Issue 4) pp:771-777
Publication Date(Web):2007 December
DOI:10.1007/s00253-007-1216-1
An actinomycete strain IBL-14 isolated from soil by utilizing diosgenin as the sole carbon and energy source was identified as Streptomyces virginiae. S. virginiae IBL-14 can transform diosgenin to isonuatigenone. To our knowledge, this is the first reported case of producing rare nuatigenin-type spirosteroid (isonuatigenone) from pyrano-spirosteroid (diosgenin) by microbial transformation. From diosgenin to isonuatigenone, the pathway has been confirmed in this study that diosgenin was first converted to diosgenone, and then diosgenone was transformed to isonuatigenone by the C25 tertiary hydroxylation reaction. It appeared to be favorable to accumulate isonuatigenone when diosgenin was added to the onset of the stationary phase of cell growth, and the yield of isonuatigenone was about 28.4% during 48 h from 1.5 mM diosgenin. The C25 tertiary hydroxylation of diosgenone by S. virginiae IBL-14 is a novel and interesting reaction and will be a practical tool in producing natural nuatigenin-type steroids.
Co-reporter:Jing-Bo Zhao;Dong-Zhi Wei;Wang-Yu Tong
Applied Microbiology and Biotechnology 2007 Volume 76( Issue 4) pp:795-800
Publication Date(Web):2007 September
DOI:10.1007/s00253-007-1050-5
To improve the plasmid stability during the production of antihuman ovarian carcinoma × antihuman CD3 single-chain bispecific antibody (AhOC×AhCD3), the Escherichia coli BL21(DE3) host cell was optimized serially. Firstly, an isogenic recombination-deficient (recA−) derivative of BL21(DE3), namely BLR(DE3), was used as host instead of BL21(DE3). Although the segregational plasmid stability was greatly improved, AhOC×AhCD3 yield was not improved due to the severe growth inhibition of plasmid-bearing BLR(DE3) cells and the competitive plasmid instability after induction. Secondly, a mutant BLR(DE3), namely BLRM(DE3), was screened by using LB agar plates plus ampicillin and isopropyl-β-d-thiogalactopyranoside. Using this new host, growth inhibition of recombinant cells after induction was eliminated, and plasmids could be stably maintained even after long-time induction in a nonselective medium. At last, about 1.2 g/l AhOC×AhCD3, which was about thrice as much as those of recombinant BL21(DE3) and BLR(DE3) strains, was yielded.
Co-reporter:Dengchao Li;Shiwei Cheng;Yuhong Ren;Derong Zhang
Biotechnology Letters 2007 Volume 29( Issue 12) pp:1825-1830
Publication Date(Web):2007 December
DOI:10.1007/s10529-007-9480-9
A new approach has been developed for the production of enantiomerically pure (S)-β-phenylalanine (S-BPA) and (R)-β-phenylalanine in aqueous medium based on enantioselective acylation and hydrolysis properties of penicillin G acylase from Escherichia coli. The acylation reaction was highly preferential for the acylation of (R)-BPA to form N-phenylacetyl-(R)-BPA using phenylacetamide as an acyl donor, which was separated and then hydrolyzed to (R)-BPA by the same enzyme at pH 7.5. The optimal acylation reaction was at pH 10, 25°C with a 2:1 molar ratio of phenylacetamide to BPA, 8 IU ml−1 enzyme and 150 mM BPA. These resulted in a conversion of about 50% BPA; enantiomeric excess of (S)-BPA and (R)-BPA separated were 98 and 99%, respectively.
Co-reporter:Ren Ren;Zhengbing Jiang;Meiyun Liu;Xinyi Tao;Yushu Ma
Molecular Biotechnology 2007 Volume 35( Issue 2) pp:103-108
Publication Date(Web):2007 February
DOI:10.1007/BF02686102
Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.
Co-reporter:Ying Zhou;Jiashi Wei;Tao Jiang;Weifeng Gao;Yushu Ma
Annals of Microbiology 2007 Volume 57( Issue 4) pp:
Publication Date(Web):2007 December
DOI:10.1007/BF03175346
Four strains with high phenanthrene-degrading ability were isolated from petroleum badly polluted soil. The strainPseudomonas sp. ZJF08 demonstrated the highest rate of degradation (138. 1 mg·L−1·day−1) among them and degraded 97.1% of the phenanthrene in one week. The activities of two key enzymes of ZJF08, polycyclic aromatic hydrocarbon dioxygenase and catechol-2,3-oxygenase (C23O), were also assayed during the degradation of phenanthrene. Both of them reached their maximums on the 2nd day of degradation. The C23O gene (C7) ofPseudomonas sp. ZJF08 was cloned and expressed inEscherichia coli, and its gene product was purified by a Ni-NTA-agarose column. The optimum temperature for the purified C23O was 40°C at pH 7.5 and the C23O activity could be still detected when the temperature reached 70°C. The results showed that the C23O fromPseudomonas sp. strain ZJF08 exhibited better thermostability than its homologs reported.
Co-reporter:Guohua Hu, Yanhua Lu, Dongzhi Wei
Food Chemistry 2006 Volume 99(Issue 4) pp:693-697
Publication Date(Web):2006
DOI:10.1016/j.foodchem.2005.08.045
Chinese chive seeds (Allium tuberosum Rottl.) (grown in China) were investigated. Density, thousand-grain weight, and hectolitre weight of seeds were 1.27 g/cm3, 4.9 g, and 71 kg/100 l, respectively. The results showed that Chinese chive seeds contained high amounts of oil (15.8%), dietary fibre (18.2%) and crude protein (12.3%). Oil of seeds was composed of 10.1% saturated and 90.0% unsaturated fatty acids. Linoleic(69.1%) and palmitic (7.0%) were the most abundant unsaturated and saturated fatty acids, respectively. Chinese chive seeds contained 4.5 mg/kg of thiamin, 2.8 mg/kg of riboflavin and 55.1 mg/kg of niacin. The mineral contents of the seed of A. tuberosum, for iron, calcium and zinc, were 580 mg/kg, 1328 and 80.8 mg/kg, respectively. Analysis of the amino acid content of Chinese chive seed revealed that it was a rich source of the essential amino acids, isoleucine, tryptophan and lysine. The study revealed that Chinese chive seeds had high levels of nutritionally important components, such as oil, minerals and essential amino acids.
Co-reporter:R.-R. Kuang, F. Qian, Z. Li, D.-Z. Wei, Y. Tang
European Journal of Medicinal Chemistry 2006 Volume 41(Issue 4) pp:558-565
Publication Date(Web):April 2006
DOI:10.1016/j.ejmech.2006.01.017
Action mechanisms of four types of PI3Kγ inhibitors were investigated on the ligand-binding pocket (LBP) of PI3Kγ with molecular modeling method. At first five compounds whose complex structures with PI3Kγ were available experimentally were used to validate the reliability of docking program Autodock3.0. The results demonstrated that the program could reproduce the bound conformations of those compounds in crystal structures. Then the program was used to dock all the four types of PI3Kγ inhibitors into the LBP of the enzyme. The predicted activities of these compounds were in agreement with their experimental activities, and a pharmacophore model was hence derived for these compounds, which consisted of one hydrophobic portion flanked by two symmetric hydrophilic portions. Furthermore, the structure–activity relationships of PI3Kγ inhibitors were elucidated and the activity differences between them were discussed.
Co-reporter:Wang-Yu Tong;Xiang-Yang Fu;Dong-Zhi Wei;Xiang-Yang Fu;Dong-Zhi Wei;Wang-Yu Tong
Journal of Chemical Technology and Biotechnology 2006 Volume 81(Issue 12) pp:1866-1871
Publication Date(Web):6 NOV 2006
DOI:10.1002/jctb.1615
Terrific broth, a complex medium containing a high content of yeast extract, was chosen to cultivate recombinant Escherichia coli with the plasmid encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH). The volumetric yield of Trx–hPTH fusion protein in the culture with Terrific broth reached about 800 mg L−1 after optimization. It was found that high content of yeast extract in Terrific broth promoted cell growth and Trx–hPTH fusion protein production. However, the more interesting fact was confirmed that high content of yeast extract was also responsible for the high-level expression of Trx–hPTH fusion protein without specific inducer addition. Further studies indicated that the expression levels of Trx–hPTH fusion protein without specific inducer addition varied greatly with the content and the source of yeast extract contained in the media. Considering that some proteins are toxic to the host and their continuous expression may result in decreasing plasmid stability and protein yields, one should be cautious in selecting yeast extract in media for cultivating E. coli with plasmids carrying toxic genes under T7 control. Copyright © 2006 Society of Chemical Industry
Co-reporter:Yu Luo;Gang Wang;Yushu Ma
Journal of Chemical Technology and Biotechnology 2006 Volume 81(Issue 11) pp:1846-1848
Publication Date(Web):19 OCT 2006
DOI:10.1002/jctb.1602
A silica gel-based prolonged-release system for methanol was applied in biodiesel production catalyzed by lipase LipB68, instead of the traditional method of three-step addition of methanol. With the application of the prolonged-release system, the recycle times of the catalyst increased substantially from one-off usage to at least six cycles with no detectable decrease in activity, and the yield of biodiesel (94%) remained at a higher level compared with the yield of 92% with the previous method. The results showed the potential of this methanol addition method with little harm to the catalyst. Copyright © 2006 Society of Chemical Industry
Co-reporter:Yuhong Ren, Dongzhi Wei
Journal of Pharmaceutical and Biomedical Analysis 2004 Volume 36(Issue 2) pp:387-391
Publication Date(Web):29 October 2004
DOI:10.1016/j.jpba.2004.06.012
Bifunctional oligodeoxynucleotide-intercalator conjugates provide new tools for a selective control of gene expression and may have potential applications as novel therapeutic agents. An ‘in situ’ analysis method has been developed to determine intracellular levels of oligodeoxynucleotide- doxorubicin conjugate which may be used to reverse the multidrug resistance in KB-A-1 cells. A linear response over a broad concentration range (0.05–6.0 μM, r2 = 0.97) was obtained. The limit of detection and quantitation was set at 0.015 and 0.05 μM, respectively. Assay validation studies revealed that compared with HPLC, the method is rapid, high simple, high throughput and convenient for the determination of the conjugate in cells in situ. The uptake studies of the conjugate in cells using this method demonstrated that the cellular accumulation of the conjugate in KB-A-1 cells was dependent on a dynamic balance between influx and efflux processes.
Co-reporter:Dongzhi Wei;Yewang Zhang;Liu Yang
Journal of Chemical Technology and Biotechnology 2004 Volume 79(Issue 5) pp:480-485
Publication Date(Web):9 MAR 2004
DOI:10.1002/jctb.1003
Enzymatic synthesis of cefaclor was carried out with kinetic control. The product yield was improved by the continuous removal of product from the reaction mixture via complexation of cefaclor with 1-naphthol. The effects of pH and temperature on the enzymatic and complexing reactions were investigated. The efficiency of the enzymatic conversion of cefaclor in the complexing reaction was 80% under optimum reaction conditions. In situ product removal (ISPR) decreased product concentration in the bioreactor, consequently the yield of cefaclor increased from 57% (without ISPR) to 80% (with ISPR). The specially designed reactor allowed enzymatic reaction and product removal to be accomplished simultaneously, in which the productivity of cefaclor was improved to 65 g dm−3 by semi-continuous operation lasting for 55 h. Copyright © 2004 Society of Chemical Industry
Co-reporter:Chun-Lin Ye, Yan-Hua Lu, Dong-Zhi Wei
Phytochemistry 2004 Volume 65(Issue 4) pp:445-447
Publication Date(Web):February 2004
DOI:10.1016/j.phytochem.2003.11.002
Two flavonoids 3′-formyl-4′,6′-dihydroxy-2′-methoxy-5′-methylchalcone and (2S)-8-formyl-5-hydroxy-7-methoxy-6-methylflavanone together with five known compounds, were isolated from the dried buds of Cleistocalyx operculatus. Their structures were determined on the basis of spectroscopic analyses (UV, IR, EIMS, 1H, 13C NMR and HMBC).Two flavonoids and five known compounds were isolated from the buds of Cleistocalyx operculatus. The structures were elucidated by spectroscopic methods, including analysis by 2D NMR spectroscopy.
Co-reporter:Wu Su, Zhiyuan Chang, Keliang Gao, Dongzhi Wei
Tetrahedron: Asymmetry 2004 Volume 15(Issue 8) pp:1275-1277
Publication Date(Web):19 April 2004
DOI:10.1016/j.tetasy.2004.03.009
Gluconobacter oxydans DSM 2003 was firstly used in the production of (R)-2-hydroxy-propionic acid through microbial oxidation of racemic 1,2-propanediol. The biotransformation was processed with high enantiomeric excess (>99%) and near theoretical yield (48% of racemic 1,2-propanediol) when the substrate concentration was lower than 20 g/L. When the substrate concentration was increased, maintaining the pH at 6.0 helped to improve the enantioselectivity.Graphic(R)-2-Hydroxy-propionic acidC3H6O3Ee = 100%[α]20D=−1.8 (c 8, H2O)Source of chirality: asymmetric oxidationAbsolute configuration: R
Co-reporter:Dong-Zhi Wei;Liu Yang
Journal of Chemical Technology and Biotechnology 2003 Volume 78(Issue 4) pp:431-436
Publication Date(Web):19 MAR 2003
DOI:10.1002/jctb.749
The effects of organic cosolvents on the synthesis of ampicillin from phenylglycine methyl ester (PGME) and 6-amino penicillanic acid (6-APA) using immobilized Bacillus megaterium penicillin G acylase have been examined. Several cosolvents were tested for their influence on the enzyme in terms of enzyme stability and hydrophobicity. Among the cosolvents tested, ethylene glycol was found to increase the yield of ampicillin by 39–50%. The effects of ethylene glycol on the pKa of PGME, the hydrolysis of ampicillin and PGME, and synthetase/amidase and esterase/amidase ratios were also studied. Experimental data indicated that ethylene glycol inhibited more the hydrolysis of the ampicillin than the hydrolysis of the PGME and the synthetase/amidase ratio varied from 0.2 to 0.88 when the concentration (v/v) of the cosolvent increased from 0 to 40%. The enhancement of the synthesis yield was mainly caused by the reduction in the hydrolysis of acyl donor (PGME) and product (ampicillin) in the water–cosolvent system.
© 2003 Society of Chemical Industry
Co-reporter:Jian-Hang Zhu;Dong-Zhi Wei;Xue-Jun Cao;Ye-Qing Liu;Zhong-Yi Yuan
Journal of Chemical Technology and Biotechnology 2001 Volume 76(Issue 11) pp:1194-1200
Publication Date(Web):4 OCT 2001
DOI:10.1002/jctb.502
In order to develop an aqueous two-phase system (ATPS) for cephalexin synthesis with extractive bioconversion, the partitioning behaviour of cephalexin and 7-aminodeacetoxicephalosporanic acid (7-ADCA) in poly(ethylene glycol) (PEG)/salt ATPS were examined. Parameters such as PEG size, salt type and tie line length were investigated to find a primary extraction system. In PEG400/ammonium sulfate and PEG400/magnesium sulfate systems, the partition coefficient of cephalexin (KC) was larger than 1 while that of 7-ADCA (KA) deviated about 1.5. Addition of neutral salts, surfactants and water-miscible solvents were also investigated in the primary ATPS in order to improve the separation efficiency. KC greatly increased when neutral salts and surfactants were added to the PEG400/ammonium sulfate primary systems whereas KA was only slightly higher than that of the additive-free ATPS. In an improved ATPS for extractive bioconversion, consisting of PEG400 (20% w/w), ammonium sulfate (17.5% w/w), methanol (5% w/w) and NaCl (3% w/w), a KC value of up to 15.2 was achieved; KA was 1.8; KP (partition coefficient of phenylglycine methyl ester) was 1.2 and the recovery yield of cephalexin was 94.2%. The results obtained from the extractive bioconversion of cephalexin in the improved ATPS showed that it is feasible to perform such an enzymatic process in an ATPS and the system offers the potential as a model for enzymatic synthesis of some water soluble products.
© 2001 Society of Chemical Industry
Co-reporter:Hao Jiang, Xi Shi, Shiwei Qiu, Yanfen Dong, ... Dongzhi Wei
Journal of Otology (September 2016) Volume 11(Issue 3) pp:134-137
Publication Date(Web):1 September 2016
DOI:10.1016/j.joto.2016.09.002
ObjectiveTo determine whether a new-born child from a family carrying a deafness gene needs cochlear implantation to avoid dysphonia by screening and sequencing a deafness-related gene.ResultsBoth screening and sequencing results confirmed that the new born child had a normal GJB2 gene despite the fact that she has a brother suffering from hearing loss triggered by an allelic GJB2 c.176 del 16 mutation. We cloned the GJB2 genes derived from their respective blood genomic DNA into GFP fused plasmids and transfected those plasmids into the 293T cell line to test for gene function. While the mutated GJB2 gene (GJB2 c.176 del 16) of her deaf brother was found to be unable to form the gap junction structure between two adjacent cells, the baby girl's GJB2 gene ran into no such problems.ConclusionThe screening and sequencing as well as the GJB2 gene function tests invariably showed results consistent with the ABR tested hearing phenotype, which means that the child, with a normal wild type GJB2 gene, does not need early intervention to prevent her from developing hearing loss and dysphonia at a later stage in life.
Co-reporter:Pengyong You, Erzheng Su, Xuepeng Yang, Duobin Mao, Dongzhi Wei
Journal of Molecular Catalysis B: Enzymatic (September 2011) Volume 71(Issues 3–4) pp:152-158
Publication Date(Web):1 September 2011
DOI:10.1016/j.molcatb.2011.04.012
An efficient approach to synthesize terpene esters using plant lipase-Carica papaya lipase (CPL) as the biocatalyst was developed in this work. The effects of chain length of acyl donor and solvent type on the CPL-catalyzed transesterification reaction were investigated firstly. It was found that CPL showed the highest activity in n-hexane with vinyl octanoate as the best acyl donor. To obtain high yield of terpene esters, the main reaction parameters were studied and further optimized by response surface methodology. Ping–Pong Bi–Bi mechanism with dead end complex of citronellol was found to fit the initial rate data and the kinetic parameters were obtained by regression analysis. The optimal conditions were: 55 °C, 9% (w/w) of CPL based on substrate, equimolar ratio of substrates. Under these conditions, yield of more than 99% was achieved after 8 h reaction. Ionic liquids (ILs) were used to improve the operational stability because the CPL was found to lose its activity markedly during the repeated runs, it showed that the stability of CPL increased about 5 times when it was coated with ionic liquids. The CPL is low cost yet effective, thus the process developed here shows obvious potential for the production of terpene esters industrially.Graphical abstractDownload full-size imageHighlights► Terpene esters of linalool and citronellol were successfully synthesized using Carica papaya lipase. ► The effects of chain length of acyl donor and solvent type were investigated. ► The main reaction parameters were further optimized by response surface methodology. ► Stability of lipase was increased about 5 times when it was pretreated with ionic liquids.
Co-reporter:Lujia Zhang, Bo Yin, Chao Wang, Shuiqin Jiang, Hualei Wang, Y. Adam Yuan, Dongzhi Wei
Journal of Structural Biology (November 2014) Volume 188(Issue 2) pp:93-101
Publication Date(Web):1 November 2014
DOI:10.1016/j.jsb.2014.10.003
Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys–Glu–Lys catalytic triad to hydrolyze non-peptide carbon–nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.
Co-reporter:Erzheng Su, Dongzhi Wei
Journal of Molecular Catalysis B: Enzymatic (November 2008) Volume 55(Issues 3–4) pp:118-125
Publication Date(Web):1 November 2008
DOI:10.1016/j.molcatb.2008.03.001
A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log Pinterface − log Psubstrate| and thus led to a faster reaction. The more the values of |log Pinterface − log Psubstrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.
Co-reporter:Zuanning Yuan, Bo Yin, Dongzhi Wei, Yu-ren Adam Yuan
Journal of Structural Biology (May 2013) Volume 182(Issue 2) pp:125-135
Publication Date(Web):1 May 2013
DOI:10.1016/j.jsb.2013.03.001
Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Cyanobacterium Synechococcus contains one ALDH enzyme (Sp2771), together with a novel 2-oxoglutarate decarboxylase, to complete a non-canonical tricarboxylic acid cycle. However, the molecular mechanisms for substrate selection and cofactor preference by Sp2771 are largely unknown. Here, we report crystal structures of wild type Sp2771, Sp2771 S419A mutant and ternary structure of Sp2771 C262A mutant in complex with NADP+ and SSA, as well as binary structure of Gluconobacter oxydans aldehyde dehydrogenase (Gox0499) in complex with PEG. Structural comparison of Sp2771 with Gox0499, coupled with mutational analysis, demonstrates that Ser157 residue in Sp2771 and corresponding Pro159 residue in Gox0499 play critical structural roles in determining NADP+ and NAD+ preference for Sp2771 and Gox0499, respectively, whereas size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection. Hence, our work has provided insightful structural information into cofactor and substrate selection by ALDH.
Co-reporter:Rong Chen, Xu Liu, Jiale Wang, Jinping Lin, Dongzhi Wei
Enzyme and Microbial Technology (March 2015) Volume 70() pp:18-27
Publication Date(Web):1 March 2015
DOI:10.1016/j.enzmictec.2014.12.004
•The GoKR catalyzed the asymmetric reduction with anti-Prelog stereoselectivity.•The GoKR produced chiral CHBE and HPBE with excellent e.e. (>99%).•The mechanism of anti-Prelog stereoselective reduction was analyzed by docking.A new anti-Prelog stereospecific carbonyl reductase (GoKR) from Gluconobacter oxydans DSM2343 was cloned and identified in Escherichia coli. This GoKR formed a homo-tetramer with a subunit size of approximately 27.0 kDa. GoKR exhibited full activity with NADPH but not with NADH as a cofactor. The optimal pH and temperature were 9.0 and 30 °C, respectively. GoKR reduced various ketones, including aliphatic and aromatic ketones, α- and β-keto esters. Aromatic ketones were reduced to (R)-enantiomers, whereas keto esters were reduced to (S)-hydroxy esters with different enantioselectivities. The data indicate that GoKR does not obey Prelog's rule and exhibits anti-Prelog enantiopreference. Enzyme–substrate–cofactor docking analysis showed that hydride transfer occurred at the si faces of carbonyl group for ethyl 4-chloro-3-oxobutanoate (COBE), which was then selectively reduced to the chiral (S)-alcohol. Excellent enantioselectivities were obtained for reducing COBE and ethyl 2-oxo-4-phenylbutyrate into the corresponding (S)-type products. These products are important for synthesizing HMG-CoA reductase (statins) and angiotensin-converting enzyme inhibitors, respectively.
Co-reporter:Bei Gao, Tao Xu, Jinping Lin, Lujia Zhang, Erzheng Su, Zhengbing Jiang, Dongzhi Wei
Journal of Molecular Catalysis B: Enzymatic (March 2011) Volume 68(Issues 3–4) pp:286-291
Publication Date(Web):1 March 2011
DOI:10.1016/j.molcatb.2010.12.001
The capacity of lipase LipK107 from Proteus sp. catalyzing the kinetic resolution of racemates was investigated. The resolution of racemic 1-phenylethanol in organic medium was selected as model reaction. The conversion was dramatically dependent on the water content and the LipK107 showed high activity in a wide range of water content without appreciable loss of enzyme enantiodiscrimination. Besides, the chain length of acyl donor also had a significant effect on the conversion, and the highest enantioselectivity was achieved when methyl palmitate was used. Based on the analysis of computer model structure of LipK107, different mutations were introduced into the lid region. Each derivative of LipK107 was expressed, purified, and assessed of the activity. According to the prediction, using mutants E130L + K131I and T138V as catalyst, respectively, the conversions of 1-phenylethanol improved greatly with a slight increase of enantiodiscrimination. In addition, the effects of hydrophobicity and electrostatic of the lid on lipase activity were determined. This work indicated that the modification of the lid might considerably enhance the activity and improve the yield of catalytic reactions, which could apply to other lipases. The computer simulations would make the process of identifying amino acids for substitution efficiently.Graphical abstractDownload full-size imageResearch highlights▶ Based on analysis of computer model structure of LipK107, mutations were introduced. ▶ The activity of LipK107 enhanced dramatically according to the prediction. ▶ Effects of hydrophobicity and electrostatic of lid on lipase activity were determined. ▶ The modification of the lid could considerably enhance the lipase activity.
Co-reporter:Tao Xu, Lujia Zhang, Erzheng Su, Dongbing Cui, Xuedong Wang, Dongzhi Wei
Journal of Molecular Catalysis B: Enzymatic (March 2010) Volume 62(Issues 3–4) pp:288-296
Publication Date(Web):1 March 2010
DOI:10.1016/j.molcatb.2009.11.011
In the context of specifying the origin of enzyme enantioselectivity, the present study explores the lipase enantioselectivity towards secondary alcohols of similar structure from the perspective of substrate binding. By carrying out molecular mechanics minimization as well as molecular dynamics simulation on tetrahedral reaction intermediates which are used as a model of transition state, we identify an unconventional productive binding mode (PBM)—M/H permutation type for Candida antarctica lipase B (CALB). The in silico results also indicate that different PBMs of the slow-reacting enantiomer do exist in one lipase even when there is little structural differences between substrates, e.g. compounds with Ph or CH2CH2Ph group display the M/H permutation type PBM while molecules with CH2Ph show the M/L permutation type PBM. By relating the PBMs of substrates to the experimentally determined E-values obtained by Hoff et al. [16], we find that disparity in PBM of the slow-reacting enantiomer determines why E-values of substrates with CH2Ph were lower than E-values of substrates with Ph or CH2CH2Ph group. The modeling results also suggest that the “pushed aside” effect of the F atom and Br atom accommodates the medium size substituent of the substrate better in the stereospecificity pocket of the enzyme.
Co-reporter:Dandan Lv, Wei Wang, Dongzhi Wei
Plasmid (January 2012) Volume 67(Issue 1) pp:67-71
Publication Date(Web):1 January 2012
DOI:10.1016/j.plasmid.2011.10.002
We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.Highlights► Two filamentous fungi Trichoderma reesei expression vectors pWEF31 and pWEF32 were newly constructed by us. ► Both vectors was confirmed by detecting the expression of DsRed2 gene in T. reesei Rut C30. ► The two vectors will be useful for large-scale gene expression industry.
Co-reporter:Qingbao DING, Ling OU, Dongzhi WEI, Xiaokun WEI, Yanmei XU, Chunyan ZHANG
Chinese Journal of Chemical Engineering (February 2011) Volume 19(Issue 1) pp:122-127
Publication Date(Web):1 February 2011
DOI:10.1016/S1004-9541(09)60187-7
Recombinant Escherichia coli pUDP, which overexpressed uridine phosphorylase (UPase), was constructed. 0.5 mmol·L−1 lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth. The lactose-induced UPase was stable at 50 °C. Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L−1 uridine and 5-fluorouracil in phosphate buffer (pH 7.0) catalyzed at 50 °C for 1.5 h and the yield of 5-fluorouridine was higher than 68%. Under the optimum reaction conditions for production of 5-fluorouridine, 5-methyluridine and azauridine were synthesized from uridine by pUDP, the yield was 61.7% and 47.2% respectively. Deoxynucleosides were also synthesized by pUDP, but the yield was only about 20%.
Co-reporter:Xiaoqiang Ma, Erzheng Su, Ying Zhu, Senwen Deng, Dongzhi Wei
Journal of Biotechnology (December 2013) Volume 168(Issue 4) pp:607-615
Publication Date(Web):1 December 2013
DOI:10.1016/j.jbiotec.2013.08.024
•The solubility of GLA expression in E. coli was significantly affected by expression system.•Lactose used as inducer and lower inducing temperature could reduce the GLA inclusion formation.•Dextrin used as carbon source could promote the accumulation of soluble GLA in E. coli.•High-level expression of NK703 GLA was obtained with a synthetic medium and a feeding medium.In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a + GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25 °C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 U L−1 h−1 and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406 ± 521 U/L, which was the highest report at fermenter level.
Co-reporter:Tao Xu, Bei Gao, Lujia Zhang, Jingpin Lin, Xuedong Wang, Dongzhi Wei
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics (December 2010) Volume 1804(Issue 12) pp:
Publication Date(Web):December 2010
DOI:10.1016/j.bbapap.2010.08.012
In order to fully explore the structure–function relationship of a Proteus lipase (LipK107) that was screened from the soil in our previous study, we have modeled the three-dimensional (3-D) structures of the enzyme in its active and inactive conformations on the basis of crystal structures of Burkholderia glumae and Pseudomonas aeruginosa lipases in the present study. Both homology models suggested that LipK107 possessed a catalytic triad (Ser79–Asp232–H254), an oxyanion hole (Leu13 and Gln80) which was used to stabilize the reaction tetrahedral intermediates, and a lid substructure that controlled the access of the substrate to the active site. The existence of the lid was further verified by carrying out the interfacial activation experiment. The conformational change of LipK107 which was caused by lid opening action was predicted by superimposing the two theoretical models for the first time. Finally, both 3-D structures were used to predict the enantioselectivity of LipK107 when the enzyme was used to catalyze the resolution of racemic 1-phenylethanol. Lid-open model of LipK107 identified the R-enantiomer as the preferred enantiomer, while lid-closed mode showed that the S-enantiomer was more favored. However, only the lid-open conformational model could led to predictions that agreed with the following the experimental result of real biocatalysis reaction of 1-phenylethanol.Research Highlights►Contrary to other modeling researches, two models (lid-open and lid-closed conformation) of LipK107 were built, and both models identified the same key structural features of the lipase. This guaranteed the accuracy modeling results. ►In contrast to other modeling papers, experimental means were applied to verify structural features (the lid substructure) identified by homology models. ►Superimposing the two static models predicted a dynamic view of lid opening action. ►Both models were used to predict the enantioselectivity of the lipase by molecular dynamics simulation, and the predicting results were in agreement with the experimental results. ►LipK107 was originally discovered by our group. Gao et al, J Biotech. 139 (2009) 169-175.
Co-reporter:Jian-Min Song, Dong-Zhi Wei
Biomass and Bioenergy (December 2010) Volume 34(Issue 12) pp:
Publication Date(Web):1 December 2010
DOI:10.1016/j.biombioe.2010.08.010
Bagasse, the main coproduct in the sugar industry, has been shown to be the sole carbon source for liquid-state fermentation to produce hydrolytic enzymes (cellulases and xylanases) using Cellulosimicrobium cellulans in flask during 3 days and the enzymatic extracts obtained at different time intervals were analysed. The degradation of bagasse indicate the lignocellulolytic capacity of C. cellulans, 26% decrease in hemicellulose content at 96 h, while 10% decrease in cellulose. This accord to that xylanases fairly predominated over cellulases. A rapid rise in the activity of cellulases and xylanases was observed in the early stages of incubation (within the first 24 h). The optimum pH for these enzymes by C. cellulans in liquid cultures was between 5.0 and 5.6 and the optimum temperature was 50 °C. Thermal stability of the enzymes was appreciable up to 45 °C, pH stability at pH 5.0.
Co-reporter:Jian Deng, Kangling Chen, Zhiqiang Yao, Jinping Lin, Dongzhi Wei
Journal of Molecular Catalysis B: Enzymatic (August 2015) Volume 118() pp:1-7
Publication Date(Web):1 August 2015
DOI:10.1016/j.molcatb.2015.04.014
•GoCR from G. oxydans could catalyze the reduction of halogenated acetophenones.•Only S-configuration alcohols (>99% ee) were produced.•500 mM o-chloroacetophenone was reduced at 96% conversion.•For o-chloroacetophenone reduction, 2.0 eq. of isopropanol served as co-substrate.•Molecular docking explained the mechanism of the high enantioselectivity of GoCR.A carbonyl reductase from Gluconobacter oxydans (GoCR) exhibited good activity and strict enantioselectivity for the asymmetric reduction of a series of halogenated acetophenones, resulting in the corresponding S-alcohols. For efficient synthesis of alcohol products, an economical and satisfactory substrate-coupled cofactor regeneration system was constructed employing isopropanol as the co-substrate to regenerate NADH in situ. In the presence of 2.0 molar eq. of isopropanol, 500 mM o-chloroacetophenone was reduced to (S)-1-(2-chlorophenyl)-ethanol with >99% ee and a high conversion rate of 96% by recombinant Escherichia coli BL21 cells overexpressing GoCR. In this reaction system, a slight excess of isopropanol as hydride source could drive the reduction reaction to near completion due to the speculated forming of an intramolecular hydrogen bond between the Cl group and the new obtained alcohol moiety of (S)-1-(2-chlorophenyl)-ethanol. Furthermore, other tested halogenated acetophenones at 100 or 200 mM substrate load were also reduced at 71–96% conversion, affording S-configuration alcohols with >99% ee. Homology modeling and molecular dynamics simulation were performed to uncover the structural basis for the excellent enantioselectivity of GoCR toward halogenated acetophenones. These results imply the high potential of GoCR in the production of halogenated 1-phenylethanols, such as (S)-1-(2-chlorophenyl)-ethanol, an important chiral building block with wide application in pharmaceutical chemistry and fine chemicals.Download full-size image
Co-reporter:Wei Wang, Xiang-Yu Shi, Dong-Zhi Wei
Journal of Microbiological Methods (August 2014) Volume 103() pp:37-39
Publication Date(Web):1 August 2014
DOI:10.1016/j.mimet.2014.05.017
•For the first time, we describe a light-mediated system in Trichoderma reesei.•Our light-mediated system is orthogonal to internal cellulase system of T. reesei.•Our light-mediated system is useful in controlling gene expression in T. reesei.We developed a light-mediated system based on synthetic light-switchable transactivators. The transactivators bind promoter upon blue-light exposure and rapidly initiate transcription of target transgenes in filamentous fungus Trichoderma reesei. Light is inexpensive to apply, easily delivered, and instantly removed, and thus has significant advantages over chemical inducers.
Co-reporter:Qinghai Liu, Jinping Lin, Meiyun Liu, Xinyi Tao, Dongzhi Wei, Xingyuan Ma, Shengli Yang
Protein Expression and Purification (August 2007) Volume 54(Issue 2) pp:212-219
Publication Date(Web):1 August 2007
DOI:10.1016/j.pep.2007.03.009
Human parathyroid hormone (hPTH) is a promising agent in the treatment of osteoporosis. The intact recombinant human parathyroid hormone [rhPTH(1–84)] was prepared in a large scale from Escherichia coli using a soluble fusion protein strategy. With degenerate codons, gene of hPTH(1–84) was synthesized, ligated with pET32a(+) vector, and then expressed in E. coli BL21(DE3) cells. The soluble fusion protein His6-thioredoxin-hPTH(1–84) was harvested after purification by immobilized metal affinity chromatography (IMAC). Following enterokinase cleavage, ion-exchange-chromatography (IEC) and size-exclusive-chromatography (SEC) were used, and finally, over 300 mg/l intact hPTH(1–84) with high purity up to 99% was obtained. The purified rhPTH(1–84) was confirmed by mass spectrometry and N-terminal/C-terminal amino-acid sequence analysis. Additionally, this product stimulated adenylate cyclase in Rat Osteosarcoma Cell UMR-106 at the same extent as hPTH standards, indicating that the purified rhPTH(1–84) has full biological activity. The efficient procedure for expression and purification of rhPTH(1–84) may be useful for the mass production of this important protein.
Co-reporter:Er-Zheng Su, Wei-Qin Xu, Ke-Liang Gao, Yu Zheng, Dong-Zhi Wei
Journal of Molecular Catalysis B: Enzymatic (3 September 2007) Volume 48(Issues 1–2) pp:28-32
Publication Date(Web):3 September 2007
DOI:10.1016/j.molcatb.2007.06.003
Substituting short-chained alkyl acetates for short-chained alcohols as acyl acceptors for fatty acid esters production, the negative effects of glycerol and alcohol on lipase can be eliminated. Short-chained alkyl acetates, like other short-chained esters, are also suitable solvents for seed oil extraction. Thus, methyl acetate and ethyl acetate were adopted as extraction solvents and transesterification reagents at the same time for in situ reactive extraction of Pistacia chinensis Bunge seed and Jatropha curcas L seed in this work. Fatty acid methyl esters and ethyl esters were, respectively obtained with higher yields than those achieved by conventional two-step extraction/transesterification. The improvement ranged from 5.3% to 22%. The key parameters such as solvent/seed ratio and water content were further investigated to find their effects on the in situ reactive extraction. The highest P. chinensis Bunge and J. curcas L methyl/ethyl esters could attain 92.8%, 89.5%, 86.1% and 87.2%, respectively under the optimized conditions.
Co-reporter:Kefeng Ni, Hualei Wang, Li Zhao, Minjie Zhang, Siyuan Zhang, Yuhong Ren, Dongzhi Wei
Journal of Biotechnology (20 September 2013) Volume 167(Issue 4) pp:433-440
Publication Date(Web):20 September 2013
DOI:10.1016/j.jbiotec.2013.07.024
•A facile and efficient whole-cell immobilization method was proposed.•An ethyl acetate–water biphasic system was built and optimized for production of R-MA.•As high as 1 M mandelonitrile could be enantioselectively hydrolyzed within 4 h.•The productivity of this system is the highest of published in production of R-MA by nitrilase.The recombinant Escherichia coli M15/BCJ2315 which harbored a mandelonitrilase from Burkholderia cenocepacia J2315 was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid. Furthermore, the immobilized cells were reused up to 15 cycles without any activity loss in completely hydrolyzing mandelonitrile (100 mM) within 1 h in aqueous solution. The ethyl acetate–water biphasic system was built and optimized. Under the optimal conditions, as high as 1 M mandelonitrile could be hydrolyzed within 4 h with a final yield and ee value of 99% and 95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for 6 batches, giving a final productivity (g L−1 h−1) and relative production (g g−1) of 40.9 and 38.9, respectively.
Co-reporter:Dongbing Cui, Lujiang Zhang, Zhiqiang Yao, Xu Liu, Jinping Lin, Y. Adam Yuan, Dongzhi Wei
Journal of Biotechnology (20 September 2013) Volume 167(Issue 4) pp:386-392
Publication Date(Web):20 September 2013
DOI:10.1016/j.jbiotec.2013.07.029
•Novel computer-based method proposed to determine and design mutant site of target protein.•Successful reconstructed coenzyme specificity from NADH to NADPH.•Hydrogen bond and electrostatic interactions are key factors determining NADPH binding.•The double-mutant design has effectively achieved coenzyme specificity alteration but without enzymatic activity change.•Theoretical guidance suggested for coenzyme specificity alteration.Short-chain dehydrogenase Gox2181 from Gluconobacter oxydans catalyzes the reduction of 2,3-pentanedione by using NADH as the physiological electron donor. To realize its synthetic biological application for coenzyme recycling use, computational design and site-directed mutagenesis have been used to engineer Gox2181 to utilize not only NADH but also NADPH as the electron donor. Single and double mutations at residues Q20 and D43 were made in a recombinant expression system that corresponded to Gox2181-D43Q and Gox2181-Q20R&D43Q, respectively. The design of mutant Q20R not only resolved the hydrogen bond interaction and electrostatic interaction between R and 2′-phosphate of NADPH, but also could enhance the binding with 2′-phophated of NADPH by combining with D43Q. Molecular dynamics simulation has been carried out to testify the hydrogen bond interactions between mutation sites and 2′-phosphate of NADPH. Steady-state turnover measurement results indicated that Gox2181-D43Q could use both NADH and NADPH as its coenzyme, and so could Gox2181-Q20R&D43Q. Meanwhile, compared to the wild-type enzyme, Gox2181-D43Q exhibited dramatically reduced enzymatic activity while Gox2181-Q20R&D43Q successfully retained the majority of enzymatic activity.
Co-reporter:Xin Gao, Kefeng Ni, Chengcheng Zhao, Yuhong Ren, Dongzhi Wei
Journal of Biotechnology (20 October 2014) Volume 188() pp:36-41
Publication Date(Web):20 October 2014
DOI:10.1016/j.jbiotec.2014.07.443
•A novel enzyme-orientated immobilization strategy was developed.•The activity recovery of immobilized FDH was improved by three folds.•The catalytic efficiency of co-immobilized FDH and LDH was increased by four folds.Immobilization of enzymes onto nanoparticles and retention of their structure and activity, which may be related to the orientation of enzymes on nanoparticles, remain a challenge. Here, we developed a novel enzyme-orientation strategy to enhance the activity of formate dehydrogenase immobilized on polydopamine-coated iron oxide nanoparticles via site-directed mutation. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on polydopamine-coated iron oxide nanoparticles to investigate the influence of these mutations on immobilization. The immobilized mutant C242A/C275V/C363V/K389C demonstrated the highest immobilization yield and retained 90% of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of l-tert-leucine. The catalytic efficiency of the co-immobilized mutant C242A/C275V/C363V/K389C and leucine dehydrogenase increased by more than 4-fold compared to that of co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase.
Co-reporter:Xin-Bin Liu, Min Liu, Xin-Yi Tao, Zhong-Xi Zhang, Feng-Qing Wang, Dong-Zhi Wei
Journal of Biotechnology (20 December 2015) Volume 216() pp:47-55
Publication Date(Web):20 December 2015
DOI:10.1016/j.jbiotec.2015.10.005
•Increasing supply and reducing competitive consumption of 2,3-oxidosqualene was used.•A marker-recycling and gene-targeting Cre-lox71/66 system was developed herein.•The yield of dammarenediol-II in P. pastoris was increased significantly.•This study constitutes a good platform for production of ginsenosides in Pichia.Dammarenediol-II is the nucleus of dammarane-type ginsenosides, which are a group of active triterpenoids exhibiting various pharmacological activities. Based on the native triterpene synthetic pathway, a dammarenediol-II synthetic pathway was established in Pichia pastoris by introducing a dammarenediol-II synthase gene (PgDDS) from Panax ginseng, which is responsible for the cyclization of 2,3-oxidosqualene to dammarenediol-II in this study. To enhance productivity, a strategy of “increasing supply and reducing competitive consumption of 2,3-oxidosqualene” was used. To increase the supply of 2,3-oxidosqualene, we augmented expression of the ERG1 gene, which is responsible for 2,3-oxidosqualene synthesis. This significantly improved the yield of dammarenediol-II over 6.7-fold, from 0.030 mg/g dry cell weight (DCW) to 0.203 mg/g DCW. Subsequently, to reduce competition for 2,3-oxidosqualene from ergosterol biosynthesis without affecting the normal growth of P. pastoris, we targeted the ERG7gene, which is responsible for conversion of 2,3-oxidosqualene to lanosterol. This gene was downregulated by replacing its native promoter with a thiamine-repressible promoter, using a marker-recycling and gene-targeting Cre- lox71/66 system developed for P. pastoris herein. The yield of dammarenediol-II was further increased more than 3.6-fold, to 0.736 mg/g DCW. Furthermore, the direct addition of 0.5 g/L squalene into the culture medium further enhanced the yield of dammarenediol-II to 1.073 mg/g DCW, which was 37.5-fold higher than the yield from the strain with the PgDDS gene introduction only. The P. pastoris strains engineered in this study constitute a good platform for further production of ginsenosides in Pichia species.
Co-reporter:Chenxi Ning, Erzheng Su, Yanjun Tian, Dongzhi Wei
Journal of Biotechnology (20 August 2014) Volume 184() pp:7-10
Publication Date(Web):20 August 2014
DOI:10.1016/j.jbiotec.2014.05.004
•Combi-CLEAs had been demonstrated to be a robust regeneration system for cofactor.•Negligible amount of cofactor successfully started high speed regeneration.•Possible homogeneous and adjacent dispersion of two enzymes may lead to quick uptake of cofactor.An alternative strategy for cofactor regeneration in the synthesis of valuable chiral alcohols catalyzed by ketoreductases was developed. combi-CLEAs of ketoreductase and d-glucose dehydrogenase enabled the repeated and effective conversion of substrate ethyl 4-chloro-3-oxobutanoate (COBE) with several superiorities. Wide application of this strategy in production of various chiral alcohols could be expected in the future for its high efficiency with low cost.Download full-size image
Co-reporter:Kun Chen, Wang-Yu Tong, Dong-Zhi Wei, Wei Jiang
Enzyme and Microbial Technology (2 July 2007) Volume 41(Issues 1–2) pp:71-79
Publication Date(Web):2 July 2007
DOI:10.1016/j.enzmictec.2006.12.002
The 11β-hydroxylation product of 16,17α-epoxyprogesterone (EP), 11β-hydroxy-16,17α-epoxyprogesterone (HEP), is an important intermediate for many anti-inflammatory drugs. To understand the hydroxylation characteristics of the 11β-hydroxylase from filamentous fungus Absidia coerulea IBL02, the 11β-hydroxylation process, the purification results and some characteristics of the enzyme (protein P450) from mycelia were first reported in this paper. When the feeding time of the substrate was set at 27 h and the biotransformation time was for 42 h after fermentation optimization in the whole-cell bioconversion, a satisfied yield of about 4 g/l with above 85% biotransformation rate was achieved. We found the 11β-hydroxylase biosynthesis in cell cultivation could be induced by adding analogs of the substrate, and an about three-fold increase in enzymatic amount was achieved by adding progesterone as inducer for about 4 h. Experimental results showed that Ca2+ and Fe2+ could greatly enhance the enzymatic activity, but Mg2+, Mn2+, Cu2+, and Zn2+ could inhibit it. After the optimization of the purification conditions, the 11β-hydroxylase (protein P450) with a single band on SDS-PAGE was obtained and estimated to be a MW of about 60 kDa, giving a maximum absorbance at 448 nm in reduced carbon monoxide difference spectra.
Co-reporter:Shiwei Cheng, Qingxun Song, Dongzhi Wei, Bingxue Gao
Enzyme and Microbial Technology (2 August 2007) Volume 41(Issue 3) pp:326-330
Publication Date(Web):2 August 2007
DOI:10.1016/j.enzmictec.2007.02.011
Different carbon sources were investigated for overproduction penicillin G acylase from Alcaligenes faecalis in recombinant Escherichia coli strains. The results indicated that the enzyme was optimally produced with 45 g/l of dextrin, and about 43,385 and 79,880 U/l for the highest enzyme activities were obtained in batch cultivations of shaken flasks and a 3.7 l bioreactor, respectively. Active site titration and SDS-PAGE electrophoretic analysis demonstrated that the maximum yield of the active enzyme was 2.54 g/l, which was about 40% of total soluble proteins. The highest specific activity of A. faecalis penicillin G acylase obtained was above 10 U/mg protein, and there was almost no plasmid lost in the whole batch cultivations. Furthermore, the cultivation process was relatively simple and suitable for large-scale production.
Co-reporter:Pixiang Wang, Xiangyu Gong, Erzheng Su, Jingli Xie, Dongzhi Wei
Biochemical Engineering Journal (15 September 2011) Volume 56(Issues 1–2) pp:17-22
Publication Date(Web):15 September 2011
DOI:10.1016/j.bej.2011.04.011
A facile pretreatment method was developed to obtain high specific activity of PGA from recombinant E. coli cells for preparing immobilized enzyme with high activity, which coupled selective extraction of PGA with butyl acetate and adsorption of butyl acetate with active carbon. Butyl acetate (5%, v/v) led to a 92.0% release of PGA, the specific activity of which in the extracting solution was twice that obtained by sonication. The negative effect on PGA immobilization due to residual butyl acetate in the extracting solution was removed by adding active carbon (8%, w/v). The final PGA solution meets requirement for industrial immobilization while eliminating the high cost of a traditional purification process. This pretreatment method developed in this work is simple, highly performing and cost-effective. It has been employed in a 10 tons/year immobilized PGA production line.
Co-reporter:Xue-Peng Yang, Liu-Jing Wei, Jian-Bin Ye, Bo Yin, Dong-Zhi Wei
Archives of Biochemistry and Biophysics (15 September 2008) Volume 477(Issue 2) pp:
Publication Date(Web):15 September 2008
DOI:10.1016/j.abb.2008.03.030
A membrane-bound pyrroloquinoline quinine (PQQ)-dependent d-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of d-sorbitol to l-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by d-sorbitol), while products are released in sequence (first l-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for d-sorbitol, in addition to PQQ-binding and Mg2+-binding sites in the catalytic center.
Co-reporter:Xiaoqiang Ma, Senwen Deng, Erzheng Su, Dongzhi Wei
Biochemical Engineering Journal (15 March 2015) Volume 95() pp:1-8
Publication Date(Web):15 March 2015
DOI:10.1016/j.bej.2014.11.015
•One-pot bi-enzymatic process was developed for production of D-7-ACA from CPC.•Higher D-7-ACA yield was achieved in 30 min under the optimized enzyme amount.•The specific productivity of D-7-ACA was about 4 times higher than that of two-pot process.•This one-pot process also showed good operational stability.The three-enzyme three-pot using immobilized d-amino acid oxidase (DAAO), glutaryl-7-ACA acylase (GLA), and cephalosporin C deacetylase (CAH) was the main strategy for industrial conversion of cephalosporin C (CPC) to deacetyl-7-aminocephalosporanic acid (D-7-ACA), a key pharmaceutical intermediate that is used for the production of 3-vinyl substituted β-lactams antibiotics. In order to simplify this traditional process, we developed an one-pot bi-enzyme catalyzed procedure by coupling immobilized cephalosporin C acylase (CPCA) with immobilized cephalosporin C deacetylase (CAH), which contained two parallel cascade reactions: firstly, hydrolysis of CPC to 7-ACA by CPCA, then deacetylation of 7-ACA to D-7-ACA by CAH (route 1); or firstly, hydrolysis of CPC to deacetyl-CPC (D-CPC) by CAH, then de-α-aminoadipoylation of D-CPC to D-7-ACA by CPCA (route 2). A D-7-ACA yield of 78.39% was achieved in 30 min in a single reactor under the optimized enzyme amount. The specific productivity of D-7-ACA reached 10.85 g g−1 h−1 L−1, which increased approximately 4-fold compared to that obtained from the two-pot enzymatic process. The half-time of immobilized CPCA was approximately 18 h while immobilized CAH was approximately 534 h under the operational conditions. These results demonstrated the industrial feasibility of this one-pot process and its clear advantages towards the traditional three-enzyme three-pot process.Download full-size image
Co-reporter:Bei Gao, Erzheng Su, Jinping Lin, Zhengbing Jiang, Yushu Ma, Dongzhi Wei
Journal of Biotechnology (15 January 2009) Volume 139(Issue 2) pp:169-175
Publication Date(Web):15 January 2009
DOI:10.1016/j.jbiotec.2008.10.004
A lipase-producing bacterium K107 was isolated from soil samples of China and identified to be a strain of Proteus sp. With genome-walking method, the open reading frame of lipase gene lipK107, encoding 287 amino acids, was cloned and expressed in a heterologous host, Escherichia coli BL21 (DE3). The recombinant lipase was purified and characterized, and the optimum pH of the purified LipK107 was 9, at 35 °C. The recombinant E. coli expressing lipK107 was applied in biodiesel production in the form of whole-cell biocatalyst. Activity of the biocatalyst increased significantly when cells were permeabilized with 0.3% (w/v) cetyl-trimethylammoniumbromide (CTAB). This transesterification was carried out efficiently in a mixture containing 5 M equivalents of methanol to the oil and 100% water by weight of the substrate. It was the first time to use E. coli whole-cell biocatalyst expressing lipase in biodiesel production, and the biodiesel reached a yield of nearly 100% after 12 h reaction at the optimal temperature of 15 °C, which was the lowest temperature among all the known catalyst in biodiesel production.
Co-reporter:Haiyi Zhang, Yu Zheng, Qinghai Liu, Xinyi Tao, Wenyun Zheng, Xingyuan Ma, Dongzhi Wei
Biochemical Engineering Journal (15 February 2009) Volume 43(Issue 2) pp:163-168
Publication Date(Web):15 February 2009
DOI:10.1016/j.bej.2008.09.013
Co-reporter:Chenxi Ning, Erzheng Su, Dongzhi Wei
Archives of Biochemistry and Biophysics (15 December 2014) Volume 564() pp:219-228
Publication Date(Web):15 December 2014
DOI:10.1016/j.abb.2014.10.007
Co-reporter:Dongbing Cui, Lujia Zhang, Zhiqiang Yao, Xu Liu, Jinping Lin, Y.Adam Yuan, Dongzhi Wei
Journal of Biotechnology (10 May 2016) Volume 225() pp:
Publication Date(Web):10 May 2016
DOI:10.1016/j.jbiotec.2016.01.034
Co-reporter:Jian Deng, Zhiqiang Yao, Kangling Chen, Y. Adam Yuan, Jinping Lin, Dongzhi Wei
Journal of Biotechnology (10 January 2016) Volume 217() pp:31-40
Publication Date(Web):10 January 2016
DOI:10.1016/j.jbiotec.2015.11.003
•Crystal structure of carbonyl reductase GoCR was determined at high resolution.•Substrate-dependent enantioselectivity of GoCR was elucidated combining docking with MD.•GoCR was engineered with enhanced or inverted enantioselectivity towards the reduction of OPBE.•Mutant W193A was demonstrated to be a promising biocatalyst for the production of R-HPBE.In our previous work, a NAD(H)-dependent carbonyl reductase (GoCR) was identified from Gluconobacter oxydans, which showed moderate to high enantiospecificity for the reduction of different kinds of prochiral ketones. In the present study, the crystal structure of GoCR was determined at 1.65 Å resolution, and a computational strategy concerning substrate-enzyme docking and all-atom molecular dynamics (MD) simulation was established to help understand the molecular basis of enantiopreference and enantiorecognition for GoCR, and to further guide the design and engineering of GoCR enantioselectivity. For the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), three binding pocket residues, Cys93, Tyr149, and Trp193 were predicted to play a critical role in determining the enantioselectivity. Through site-directed mutagenesis, single-point mutant W193A was constructed and proved to reduce OPBE to ethyl (R)-2-hydroxy-4-phenylbutyrate (R-HPBE) with a significantly improved ee of >99% compared to 43.2% for the wild type (WT). Furthermore, double mutant C93 V/Y149A was proved to even invert the enantioselectivity of GoCR to afford S-HPBE at 79.8% ee.
Co-reporter:Senwen Deng, Xiaoqiang Ma, Erzheng Su, Dongzhi Wei
Journal of Biotechnology (10 February 2016) Volume 219() pp:142-148
Publication Date(Web):10 February 2016
DOI:10.1016/j.jbiotec.2015.12.034
•Cascade synthesis of ampicillin from penicillin G without isolation of intermediate 6-APA.•Cascade synthesis of ampicillin improved by mutant PGA.•An ampicillin yield of 87% was achieved in aqueous medium.•Less than 1.7 mole acyl donor d-PGME were required to produce 1 mole ampicillin.To avoid isolation and purification of the intermediate 6-aminopenicillanic acid (6-APA), a two-enzyme two-step cascade synthesis of ampicillin from penicillin G was established. In purely aqueous medium, penicillin G hydrolysis and ampicillin synthesis were catalyzed by immobilized wild-type and mutagenized penicillin G acylases from Alcaligenes faecalis (Af PGA), respectively (Fig. 1). The βF24 G mutant Af PGA (the 24th Phenylalanine of the β-subunit was replaced by Glycine) was employed for its superior performance in enzymatic synthesis of ampicillin. By optimizing the reaction conditions, including enzyme loading, temperature, initial pH and d-PGME/6-APA ratio, the conversion of the second step of ampicillin synthesis reached approximately 90% in 240 min and less than 1.7 mole d-PGME were required to produce 1 mole ampicillin. Overall, in a 285 min continuous two-step procedure, an ampicillin yield of 87% was achieved, demonstrating the possibility of improving the cascade synthesis of ampicillin by mutagenized PGA, providing an economically efficient and environmentally benign procedure for semi-synthetic penicillins antibiotics synthesis.Download high-res image (116KB)Download full-size image
Co-reporter:Senwen Deng, Erzheng Su, Xiaoqiang Ma, Shengli Yang, Dongzhi Wei
Journal of Biotechnology (10 April 2015) Volume 199() pp:62-68
Publication Date(Web):10 April 2015
DOI:10.1016/j.jbiotec.2015.01.004
•Protein engineering was performed on thermo-stable Alcaligenes faecalis penicillin G acylase.•A screened mutant showed up to 800-fold increased catalytic efficiency for activated acyl donor.•95% of 6-APA could be converted to ampicillin in aqueous medium by mutant PGA.•Process productivity of enzymatic synthesis of ampicillin was improved by more than 130 times.Semi-synthetic β-lactam antibiotics (SSBAs) are one of the most important antibiotic families in the world market. Their enzymatic synthesis can be catalyzed by penicillin G acylases (PGAs). In this study, to improve enzymatic synthesis of ampicillin, site-saturating mutagenesis was performed on three conserved amino acid residues: βF24, αR146, and αF147 of thermo-stable penicillin G acylase from Alcaligenes faecalis (Af PGA). Four mutants βF24G, βF24A, βF24S, and βF24P were recovered by screening the mutant bank. Kinetic analysis of them showed up to 800-fold increased kcat/Km value for activated acyl donor D-phenylglycine methyl ester (D-PGME). When βF24G was used for ampicillin synthesis under kinetic control at industrially relevant conditions, 95% of nucleophile 6-aminopenicillanic acid (6-APA) was converted to ampicillin in aqueous medium at room temperature while 12% process time is needed to reach maximum product accumulation at 25% enzyme concentration compared with the wild-type Af PGA. Consequently, process productivity of enzymatic synthesis of ampicillin catalyzed by Af PGA was improved by more than 130 times, which indicated an enzyme viable for efficient SSBAs synthesis.Download full-size image
Co-reporter:Lulu Shi, Kefei Li, Huan Zhang, Xu Liu, Jinping Lin, Dongzhi Wei
Journal of Biotechnology (10 April 2014) Volume 175() pp:69-74
Publication Date(Web):10 April 2014
DOI:10.1016/j.jbiotec.2014.01.035
•We tested a novel promoter of Gluconobacter oxydans, denominated gHp0169.•Use of gHp0169 promoter for heterologous and homologous gene expression was demonstrated.•The gHp0169 promoter exhibited a higher transcriptional activity than that of tufB promoter from G. oxydans.Gluconobacter oxydans can perform rapid incomplete oxidation of many sugars, sugar polyols and alcohols, and this outstanding ability shows a great potential in industrial bioconversion. Improvements of these industrially important strains would boost their productivities of important metabolites. However, the shortage of molecular tools for homologous and heterologous gene expression has obviously hindered G. oxydans from further application. In this study, a putative promoter sequence (104 bp), designated as gHp0169, was isolated and characterized from the chromosome of G. oxydans DSM 2003. Within this promoter sequence, the typical motif, known as -35 and -10 sequences with a 19-bp spacing, was found. The availability and promoter strength of promoter gHp0169 were then evaluated, by insertion into the plasmid pBBR1MCS5 for expression of a green fluorescent protein (GFP) and a membrane-bound type II NADH dehydrogenase (NDH-2) of G. oxydans. In comparison with promoter G. oxydans_tufB, gHp0169 exhibited a stronger promoter activity of NDH-2, indicating its significant value of gene expression in G. oxydans. To promote the production of 2-keto-d-gluconic acid (2-KGA) from gluconic acid (GA) gHp0169 was attempted to equip the flavin-dependent gluconate-2-dehydrogenase (GA2DH) and successfully achieved its overexpression in G. oxydans DSM 2003. As a result, the space-time yield of 2-KGA was boosted up to 29.86 mM/h compared with 14.78 mM/h for the control, which corresponded to a yield of 98.3% (84% for control).
Co-reporter:Guodong Wei, Xuepeng Yang, Wenyu Zhou, Jinping Lin, Dongzhi Wei
Biochemical Engineering Journal (1 December 2009) Volume 47(Issues 1–3) pp:127-131
Publication Date(Web):1 December 2009
DOI:10.1016/j.bej.2009.07.016
Co-reporter:Jianguo Zhang, Xuedong Wang, Erzheng Su, Guochen Fang, Yuhong Ren, Dongzhi Wei
Biochemical Engineering Journal (1 August 2008) Volume 41(Issue 1) pp:74-78
Publication Date(Web):1 August 2008
DOI:10.1016/j.bej.2008.03.009
Co-reporter:Xin Gao, Chengcheng Zhao, Ting Yu, Shengli Yang, Yuhong Ren and Dongzhi Wei
Chemical Communications 2015 - vol. 51(Issue 50) pp:NaN10133-10133
Publication Date(Web):2015/05/14
DOI:10.1039/C5CC02544C
A strategy for constructing a reusable multi-enzyme supramolecular device was developed by reprogramming protein–protein interactions and disulfide bond locking. The resultant multi-enzyme supramolecular device demonstrated good reusability, and approximately 80% of its initial catalytic activity was retained even after eight cycles of reuse.
Co-reporter:Shuiqin Jiang, Lujia Zhang, Zhiqiang Yao, Bei Gao, Hualei Wang, Xiangzhao Mao and Dongzhi Wei
Catalysis Science & Technology (2011-Present) 2017 - vol. 7(Issue 5) pp:NaN1128-1128
Publication Date(Web):2017/02/15
DOI:10.1039/C7CY00060J
The development of robust biocatalysts producing a large range of organic amides by hydration of nitriles is an important pursuit and challenge. A nitrilase with a broad range of nitrile substrates was switched to a nitrile hydratase by rationally regulating the reaction pathways. Five mutants improved the amide formation in the product, and four of them formed >50% amide. F193N, with the highest amide formation among the four mutants, improved its amide product up to 73%, which was 35-fold that of the wild type, while maintaining 50% activity relative to the wild type. This study would afford a new synthetic route to amides from nitriles and could be a valuable addition to the synthetic repertoire. Further protein engineering may expand the reaction range of an enzyme to afford more additional pathways to synthetic biology.
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