Co-reporter:Hongli Fang, Yandong Dou, Jingyan Ge, Mohit Chhabra, Hongyan Sun, Pengfei Zhang, Yuguo Zheng, and Qing Zhu
The Journal of Organic Chemistry October 20, 2017 Volume 82(Issue 20) pp:11212-11212
Publication Date(Web):September 19, 2017
DOI:10.1021/acs.joc.7b01594
We report herein the first example of Cu(II)-catalyzed site selective azidation of aromatic amines via C–H functionalization in aqueous media. In our strategy, a mild reagent was utilized. H2O2 served as the oxidant, and sodium azide was used as the azidation reagent. This method could also be applied to late-stage functionalization of drugs that possess an aromatic amine moiety. In addition, we found that bromination or iodination on the ortho-position of aromatic amines could occur efficiently using this catalytic system.
Co-reporter:Zhi-Qiang Liu, Lin Wu, Xiao-Jian Zhang, Ya-Ping Xue, and Yu-Guo Zheng
Journal of Agricultural and Food Chemistry May 10, 2017 Volume 65(Issue 18) pp:3721-3721
Publication Date(Web):April 20, 2017
DOI:10.1021/acs.jafc.7b00866
tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and kcat/Km, respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L–1 h–1 g–1 wet cell weight. This study presents a new carbonyl reductase for efficient synthesis of (3R,5S)-CDHH.Keywords: carbonyl reductase; random mutagenesis; single batch reaction; site-saturation mutagenesis; tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate;
Co-reporter:Zhe-Ming Wu;Ren-Chao Zheng;Xiao-Ling Tang
Applied Microbiology and Biotechnology 2017 Volume 101( Issue 5) pp:1953-1964
Publication Date(Web):2017 March
DOI:10.1007/s00253-016-7921-x
Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, Km value of 24.73 mM, and kcat/Km value of 22.47 mM−1 s−1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (eep) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.
Co-reporter:Yu-Guo Zheng;Huan-Huan Yin;Dao-Fu Yu
Applied Microbiology and Biotechnology 2017 Volume 101( Issue 3) pp:987-1001
Publication Date(Web):2017 February
DOI:10.1007/s00253-016-8083-6
Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.
Co-reporter:Ya-Ping Xue;Biao Jiao;Deng-En Hua;Feng Cheng
Bioprocess and Biosystems Engineering 2017 Volume 40( Issue 10) pp:1565-1572
Publication Date(Web):07 July 2017
DOI:10.1007/s00449-017-1812-0
Arylacetonitrilases have been widely acknowledged as important alternatives to chemical catalysts for synthesizing optically pure 2-hydroxyphenylacetic acids from nitriles. In this work, two residues (Thr132 and Ser190) located at the catalytic tunnel in the active site of an arylacetonitrilase nitA from uncultured organisms were mutated separately by site-directed mutagenesis. Ser190 was demonstrated to be the critical position which has a greater influence on arylacetonitrilase nitA activity than Thr132. The replacement of serine at position 190 with glycine increases its activity toward mandelonitrile and (o, m, p)-chloromandelonitrile, whereas replacing it with leucine abolished its activity. The best mutant S190G exhibited threefold higher specific activity toward mandelonitrile compared with that of wild-type nitA, which rendered it promising for industrial application. Homology modeling and molecular docking experiments were in agreement with the kinetic assays and support the improved catalytic performance.
Co-reporter:Yuan-Shan Wang, Qi-Wei Hu, Xing-Chang Zheng, Jian-Fen Zhang, Yu-Guo Zheng
Journal of Microbiological Methods 2017 Volume 140(Volume 140) pp:
Publication Date(Web):1 September 2017
DOI:10.1016/j.mimet.2017.03.016
Co-reporter:Kun Niu;Xiu-Li Cheng;Hai-Bin Qin;Ji-Song Liu
3 Biotech 2017 Volume 7( Issue 5) pp:314
Publication Date(Web):14 September 2017
DOI:10.1007/s13205-017-0966-4
3-Hydroxypropionic acid (3-HP) is an important compound and precursor for a series of chemicals and polymeric materials. In this study, the 3-HP producing bacteria were constructed and studied for efficient synthesis of 3-HP. The results indicated that the instability of glycerol dehydratase (GDHt) affected the 3-HP production significantly, which was successfully solved by the expression of glycerol dehydratase reactivase (GdrB), with fivefold increase in 3-HP yield. Meanwhile, NAD+-regenerating enzymes GPD1 (glycerol-3-phosphate dehydrogenase) was expressed; however, the results showed 3-HP was significantly decreased from 56.73–4 mM, and malic acid was obviously increased. Analysis of the C flux distribution showed that the main reason for the results was the lack of NAD+. The addition of NAD+ further increased the 3-HP production to 23.87 mM, demonstrating that the “regeneration of NAD+” was the major factor for enhancing 3-HP production.
Co-reporter:Feng Cheng;Jian-Miao Xu;Chao Xiang;Zhi-Qiang Liu;Li-Qing Zhao
Biotechnology Letters 2017 Volume 39( Issue 4) pp:567-575
Publication Date(Web):2017 April
DOI:10.1007/s10529-016-2278-x
To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation.A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G).The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.
Co-reporter:Xia Ke, Guan-Jun Ding, Bin-Xiang Ma, Zhi-Qiang Liu, Jin-Feng Zhang, Yu-Guo Zheng
Process Biochemistry 2017 Volume 62(Volume 62) pp:
Publication Date(Web):1 November 2017
DOI:10.1016/j.procbio.2017.07.030
•A putative sterol 14α-demethylase gene from Rhodococcus triatomaeis (RtCYP51) was cloned and heterologously expressed in E. coli.•Purified RtCYP51 exhibited a typical reduced CO-difference spectrum and displayed a dissociation constant (Kd) value of 2.93 μM towards lanosterol.•Three exogenous electron donor systems were selected to couple with RtCYP51 and it presented a relatively high turnover number of 0.63 nmol lanosterol/min/nmol when coupled with Fdx-FdR.•The homology model was constructed and the key residues involved in lanosterol 14α-demethylation were predicted.Reconstitution of a selective demethylation system for lanosterol is desperately needed for more efficient synthesis of steroidal drugs. Sterol 14α-demethylase cytochrome P450 (CYP51) has been confirmed to catalyze sterol 14α-demethylation, an essential reaction in sterol biosynthesis. Herein, a putative CYP51 gene (RtCYP51) was mined from the complete genome sequence of Rhodococcus triatomae BKS 15-14. Its amino acid sequence showed 25–68% identity to other sterol 14α-demethylases, and contained a novel alanine-rich sequence at the C-terminus. Heterologous expression of the RtCYP51 gene in Escherichia coli (E. coli) yielded a ∼54 kDa recombination protein that exhibited a typical reduced CO-difference spectrum and a dissociation constant (Kd) of 2.93 μM for lanosterol. Furthermore, three exogenous electron donor systems, including Fdx-FdR (Acinetobacter sp.OC4 ferredoxin and ferredoxin reductase), Fld-FdR2 (E. coli flavodoxin and flavodoxin reductase) and NfFdR (Nocardia farcinica iron-sulfur containing NADPH-P450 reductase) were selected for coupling the electron-transfer from the coenzyme to RtCYP51. Fdx-FdR was found to be the most efficient electron donor and was also confirmed to support the lanosterol demethylation activity of RtCYP51 in vitro. Under the optimum molar ratio of RtCYP51/FdR/Fdx (1:2:10), RtCYP51 exhibited a relatively high turnover number of 0.63 min−1 (nmol metabolized lanosterol/min/nmol RtCYP51), compared with known bacterial CYP51s.Download high-res image (165KB)Download full-size image
Co-reporter:Ren-Chao Zheng;Xin-Jian Yin
Journal of Chemical Technology and Biotechnology 2016 Volume 91( Issue 5) pp:1314-1319
Publication Date(Web):
DOI:10.1002/jctb.4724
Abstract
BACKGROUND
The regioselective hydration of alicyclic α,ω-dinitrile 1-cyanocyclohexaneacetonitrile (1-CCHAN), followed by hydrogenation of ω-cyano group is a green and elegant route to gabapentin. As the selective chemical hydration of dinitrile is virtually impossible, a bioprocess for regioselective hydration of 1-CCHAN was developed using microbial nitrile hydratase.
RESULTS
A newly isolated NHase producing strain, Rhodococcus aetherivorans ZJB1208, was successfully used for hydration of 1-CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed-batch biotransformation was performed in non-buffered water system with the continuous precipitation of 1-cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L−1 (6.0 mol L−1), giving a product concentration of 966.7 g L−1 and biocatalyst yield (gproduct/gcat) of 204.2.
CONCLUSION
This study is the first example of microbial nitrile hydratase with both excellent regioselectivity and strong substrate tolerance for alicyclic dinitrile and affords a potentially industrial route to gabapentin. © 2015 Society of Chemical Industry
Co-reporter:Jian-Miao Xu, Fang-Tian Fu, Hai-Feng Hu, Yu-Guo Zheng
Analytical Biochemistry 2016 Volume 495() pp:29-31
Publication Date(Web):15 February 2016
DOI:10.1016/j.ab.2015.11.012
Abstract
A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)–calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)–calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations.
Co-reporter:Xiang Chen, Zhi-Qiang Liu, Chao-Ping Lin, Yu-Guo Zheng
Bioorganic Chemistry 2016 Volume 65() pp:82-89
Publication Date(Web):April 2016
DOI:10.1016/j.bioorg.2016.02.002
•An effective chemoenzymatic strategy was developed for (S)-duloxetine production.•Carbonyl reductase from Rhodosporidium toruloides was employed in the key step.•Cofactor regeneration was accomplished using RtSCR9 coupled glucose dehydrogenase.•Production of chiral intermediate (S)-3a with so far the highest substrate loading.•(S)-duloxetine was prepared in 60.2% yield from 2-acethylthiophene with >98.5% ee.A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000 mM) in a space-time yield per gram of biomass (DCW) of 22.9 mmol L−1 h−1 g DCW−1 at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.
Co-reporter:Bo Zhang;Zhi-Qiang Liu;Chang Liu
Biotechnology Letters 2016 Volume 38( Issue 12) pp:2153-2161
Publication Date(Web):2016 December
DOI:10.1007/s10529-016-2207-z
To construct, test and exploit the CRISPRi system for enhancement of shikimic acid production with Corynebacterium glutamicum.The CRISPRi system was used to regulate C. glutamicum gene expression at the transcriptional level. Hfq protein-mediated small regulatory RNAs system was compared with CRISPRi system. The more efficient CRISPRi system was used to adjust the metabolic flux involving the shikimic acid (SA) synthetic pathway. In 11 candidate genes, including transcription regulator, three targets were effective for increasing SA production. Through over-expression of ncgl1512 and down-regulating the expression of ncgl2008, ncgl2809, ncgl1856, the titers of SA increased 115 % to 7.76 g/l in 250 ml flasks and 23.8 g/l in 5 l fermentor, which is the highest shikimic acid yield reported for C. glutamicum.CRISPRi system was constructed and is a high-performance and time-saving method to manipulate multiple genes in C. glutamicum for shikimic acid production. Moreover, CRISPRi-system was also effective in regulating the expression of a transcription regulator.
Co-reporter:Shan Lin;Zhi-Qiang Liu;Ya-Ping Xue
Applied Biochemistry and Biotechnology 2016 Volume 179( Issue 4) pp:633-649
Publication Date(Web):2016 June
DOI:10.1007/s12010-016-2020-0
Hirsutella sinensis is considered as the only correct anamorph of Ophiocordyceps sinensis. To improve cordycepin and cordycepic acid production in H. sinensis, the biosynthetic pathways of cordycepin and cordycepic acid were predicted, and verified by cloning and expressing genes involved in these pathways, respectively. Then, 5′-nucleotidase participating in biosynthetic pathway of cordycepin, hexokinase, and glucose phosphate isomerase involved in biosynthetic pathway of cordycepic acid, were demonstrated playing important roles in the corresponding biosynthetic pathway by real-time PCR, accompanying with significantly up-regulated 15.03-, 5.27-, and 3.94-fold, respectively. Moreover, the metabolic regulation of H. sinensis was performed. As expected, cordycepin production reached 1.09 mg/g when additional substrate of 5′-nucleotidase was 4 mg/mL, resulting in an increase of 201.1 % compared with the control. In the same way, cordycepic acid production reached 26.6 and 23.4 % by adding substrate of hexokinase or glucose phosphate isomerase, leading to a rise of 77.3 and 55.1 %, respectively. To date, this is the first time to improve cordycepin and cordycepic acid production through metabolic regulation based on biosynthetic pathway analysis, and metabolic regulation is proved as a simple and effective way to enhance the output of cordycepin and cordycepic acid in submerged cultivation of H. sinensis.
Co-reporter:Zhong-Ce Hu;Li-Yuan Peng
Applied Biochemistry and Biotechnology 2016 Volume 179( Issue 7) pp:1213-1226
Publication Date(Web):2016 August
DOI:10.1007/s12010-016-2060-5
Echinocandin B belongs to lipopeptide antifungal antibiotic bearing five types of direct precursor amino acids including proline, ornithine, tyrosine, threonine, and leucine. The objective of this study is to screen over-producing mutant in order to improve echinocandin B production; a stable mutant Aspergillus nidulans ZJB12073, which can use fructose as optimal carbon source instead of expensive mannitol, was selected from thousand isolates after several cycles of UV and microwave irradiation in turn. The results showed that mutant strain ZJB12073 exhibited 1.9-fold improvement in echinocandin B production to 1656.3 ± 40.3 mg/L when compared with the parent strain. Furthermore, the effects of precursor amino acids and some chemicals on echinocandin B biosynthesis in A. nidulans were investigated, respectively. Tyrosine, leucine, and biotin were selected as key factors to optimize the medium employing uniform design method. The results showed that the optimized fermentation medium provided another 63.1 % increase to 2701.6 ± 31.7 mg/L in final echinocandin B concentration compared to that of unoptimized medium.
Co-reporter:Li-Tao Ruan;Ren-Chao Zheng
Journal of Industrial Microbiology & Biotechnology 2016 Volume 43( Issue 8) pp:1071-1083
Publication Date(Web):2016 August
DOI:10.1007/s10295-016-1786-y
A novel amidase gene (bami) was cloned from Brevibacterium epidermidis ZJB-07021 by combination of degenerate PCR and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). The deduced amino acid sequence showed low identity (≤55 %) with other reported amidases. The bami gene was overexpressed in Escherichia coli, and the resultant inclusion bodies were refolded and purified to homogeneity with a recovery of 22.6 %. Bami exhibited a broad substrate spectrum towards aliphatic, aromatic and heterocyclic amides, and showed the highest acyl transfer activity towards butyramide with specific activity of 1331.0 ± 24.0 U mg−1. Kinetic analysis demonstrated that purified Bami exhibited high catalytic efficiency (414.9 mM−1 s−1) for acyl transfer of butyramide, with turnover number (Kcat) of 3569.0 s−1. Key parameters including pH, substrate/co-substrate concentration, reaction temperature and catalyst loading were investigated and the Bami showed maximum acyl transfer activity at 50 °C, pH 7.5. Enzymatic catalysis of 200 mM butyramide with 15 μg mL−1 purified Bami was completed in 15 min with a BHA yield of 88.1 % under optimized conditions. The results demonstrated the great potential of Bami for the production of a variety of hydroxamic acids.
Co-reporter:Ya-Ping Xue;Yue-Kai Yang;Sheng-Zhi Lv
Applied Microbiology and Biotechnology 2016 Volume 100( Issue 8) pp:3421-3432
Publication Date(Web):2016 April
DOI:10.1007/s00253-016-7381-3
Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of nitrilases. Here, we review the various methodologies developed for high-throughput screening of nitrilases and focus on their advantages and limitations.
Co-reporter:Li-Qun Jin;Dong-Jing Guo;Zong-Tong Li
Journal of Industrial Microbiology & Biotechnology 2016 Volume 43( Issue 5) pp:585-593
Publication Date(Web):2016 May
DOI:10.1007/s10295-016-1747-5
In this paper, a simple and effective method using sodium metasilicate as precursor and amine as additive was first reported to immobilize recombinant nitrilase, for efficient production of 2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio) butanenitrile. High immobilization recovery of enzyme activity (above 90 %) was achieved. The immobilized enzyme displayed better thermal stability, pH stability and shelf life compared to free nitrilase. Moreover, it showed excellent reusability and could be recycled up to 16 batches without significant loss in activity. 200 mM 2-hydroxy-4-(methylthio) butanenitrile was completely converted by the immobilized enzyme within 30 min, and the accumulation amount of 2-hydroxy-4-(methylthio) butanoic acid reached 130 mmol/g of immobilized beads after 16 batches. These encouraging results demonstrated the efficiency of the new technology for nitrilase immobilization, which has great potential in preparation of 2-hydroxy-4-(methylthio) butanoic acid.
Co-reporter:Xiang Chen, Zhi-Qiang Liu, Jian-Feng Huang, Chao-Ping Lin and Yu-Guo Zheng
Chemical Communications 2015 vol. 51(Issue 61) pp:12328-12331
Publication Date(Web):22 Jun 2015
DOI:10.1039/C5CC04652A
Three short-chain alcohol dehydrogenases from Burkholderia gladioli were discovered for their great potential in the dynamic kinetic asymmetric transformation of methyl 2-benzamido-methyl-3-oxobutanoate, and their screening against varied organic solvents and substrates. This is the first report of recombinant enzymes capable of achieving this reaction with the highest enantio- and diastereo-selectivity.
Co-reporter:Ya-Ping Xue;Cheng-Ci Shi;Zhe Xu;Biao Jiao;Zhi-Qiang Liu;Jian-Feng Huang;Yin-Chu Shen
Advanced Synthesis & Catalysis 2015 Volume 357( Issue 8) pp:1741-1750
Publication Date(Web):
DOI:10.1002/adsc.201500039
Co-reporter:Feng Xue, Zhi-Qiang Liu, Nan-Wei Wan, Hang-Qin Zhu and Yu-Guo Zheng
RSC Advances 2015 vol. 5(Issue 40) pp:31525-31532
Publication Date(Web):19 Mar 2015
DOI:10.1039/C5RA02492G
The biocatalytic production of enantiopure epichlorohydrin (ECH) has been steadily attracting more attention. For industrial applications, it is important to obtain an epoxide hydrolase (EH) that possesses the desired enantioselectivity. Site-saturation and site-directed mutagenesis of the Ser207, Asn240 and Trp182 positions were used to generate variants of EH from Agromyces mediolanus ZJB120203 with enhanced enantioselectivity for the kinetic resolution of racemic ECH. The best variant, VDF (W182F/S207V/N240D), displayed a 7-fold enhanced enantioselectivity toward racemic ECH, with an increase in the enantiomeric ratio value (E value) preferring the (R)-ECH enantiomer from 12.9 of wild-type to 90.0, as well as a 1.7-fold improvement in activity. Furthermore, we successfully applied the created recombinant Escherichia coli whole cells expressing variant VDF in the kinetic resolution of racemic ECH. Enantiopure (S)-ECH could be obtained with an enantiopurity of >99% ee and a yield of 40.5% from 450 mM racemic ECH, which is better than those of other reported EHs. These results demonstrated that the EH obtained in this study could be applied for the efficient resolution of racemic ECH.
Co-reporter:Xiang Chen, Yu-Guo Zheng, Zhi-Qiang Liu, Li-Hui Sun
Journal of Chromatography B 2015 Volume 974() pp:57-64
Publication Date(Web):1 January 2015
DOI:10.1016/j.jchromb.2014.10.020
•An improved chiral HPLC method was developed for the separation of BMOB and MBHB.•This method exhibited good selectivity, stability, repeatability and high recovery.•This novel method was suitable for screening biocatalysts with activity to BMOB.•B. gladioli ZJB-12126 was indicated as a promising strain to produce (2S, 3R)-MBHB.(2S, 3R)-methyl-2-benzamidomethyl-3-hydroxybutyrate (MBHB) is a key intermediate in the synthesis of 4-aceoxyazetidinone, a building block for the production of penems and carbapenems. More attentions have been paid to screen biocatalysts achieving asymmetric preparation of (2S, 3R)-MBHB. In this study, an improved chiral high-performance liquid chromatographic (HPLC) method was developed for the stereoselective determination of 2-benzamidomethyl-3-oxobutanoate (BMOB) and MBHB, and further employed into the biotransformation of BMOB. Chiral separation was achieved within 12 min on Chiralpak AY-H column, which was faster and more suitable for screening biocatalysts exhibited reduction activity and (2S, 3R)-stereospecificity toward BMOB than on other columns. Ultimately, a new strain, Burkholderia gladioli ZJB-12126 capable of reducing BMOB to (2S, 3R)-MBHB was successfully isolated based on this newly constructed HPLC method. Samples were prepared by liquid–liquid extraction system using ethyl acetate as the extractor solvent. The extraction recoveries of BMOB and MBHB isomers ranged from 91.6 to 94.1% with relative standard deviation (RSD) below 10%. Linear calibration curves were obtained in the concentration range of 50–5000 μg/mL for both BMOB and MBHB isomers, respectively. Intra-day and inter-day precisions and accuracy were below 15% for all isomers evaluated by RSDs and relative errors (REs), respectively. This novel method was demonstrated to be suitable for assessing the biotransformation process of BMOB.
Co-reporter:Shu-Ping Zou, Miao Liu, Qiu-Liang Wang, Yan Xiong, Kun Niu, Yu-Guo Zheng, Yin-Chu Shen
Journal of Chromatography B 2015 Volumes 978–979() pp:111-117
Publication Date(Web):26 January 2015
DOI:10.1016/j.jchromb.2014.11.028
•One step isolation of echinocandin B from broth with macroporous resin.•Nine kinds of macroporous resins were tested and HP-20 was found best for echinocandin B purification.•The static equilibrium adsorption data of echinocandin B were fitted to the Langmuir isotherm.•One-step resin 3.8-fold enrichment of echinocandin B with purity 88.5%.Echinocandin B (ECB), an echinocandin type of lipopeptide antibiotic produced by Aspergillus nidulans, is a precursor for the synthesis of novel anti-fungal drug – anidulafungin. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography was established for ECB purification from Aspergillus nidulans CCTCC M 2010275 fermentation broth. Among nine macroporous resin adsorbents tested, the non-polar resin HP-20 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Langmuir equation, and the adsorption kinetic followed the pseudo-second order model. The separation parameters of ECB from broth were optimised by dynamic adsorption/desorption experiments with the column packed with HP-20 resin. Under optimal conditions, the purity increased by 3.8-fold from 23.2% in broth to 88.5% in eluent with 87.1% recovery yield by a one-step treatment. Our study provided a one-step and effective method for large-scale production of ECB, and offered references for separating other echinocandins from broth.
Co-reporter:Zhi-Qiang Liu;Ling-Mei Zhou;Peng Liu
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 21) pp:8891-8902
Publication Date(Web):2015 November
DOI:10.1007/s00253-015-6825-5
A new two-step chemo-enzymatic approach for highly efficient synthesis of all-trans-retinyl palmitate is constructed in this study. In the first step, retinyl acetate as starting material was fully hydrolyzed to retinol by potassium hydroxide. In the hydrolysis system, anhydrous ethanol was the best co-solvent to increase the solubility of retinyl acetate. The addition amounts of 5 M potassium hydroxide and anhydrous ethanol were 8 and 10 mL against 10 g retinyl acetate, respectively, and 100 % hydrolysis rate was obtained. In the second step, esterification was catalyzed by immobilized lipase on macroporous acrylic resin AB-8 using the extracted retinol and palmitic acid as substrates in non-aqueous system. After optimization, the parameters of esterification reaction were confirmed as follows: non-aqueous solvent was selected as n-hexane, washing times of extraction solution was four times, retinol concentration was 300 g/L, substrate molar ratio of retinol to palmitic acid was 1:1.1, the amount of immobilized enzyme was 10 g/L, and the esterification temperature was 30 °C. Under the optimal conditions, this protocol resulted in a 97.5 % yield of all-trans-retinyl palmitate in 700-L reactor. After purification, all-trans-retinyl palmitate was obtained with above 99 % of purity and 88 % of total recovery rate. This methodology provides a promising strategy for the large-scale production of all-trans-retinyl palmitate.
Co-reporter:Shu-Ping Zou;Wei Zhong;Chao-Jie Xia;Ya-Nan Gu
Bioprocess and Biosystems Engineering 2015 Volume 38( Issue 10) pp:1845-1854
Publication Date(Web):2015 October
DOI:10.1007/s00449-015-1425-4
A combination of microbial strain improvement and statistical optimization is investigated to maximize echinocandin B (ECB) production from Aspergillus nidulans ZJB-0817. A classical sequential mutagenesis was studied first by using physical (ultraviolet irradiation at 254 nm) and chemical mutagens (lithium chloride and sodium nitrite). Mutant strain ULN-59 exhibited 2.1-fold increase in ECB production to 1583.1 ± 40.9 mg/L when compared with the parent strain (750.8 ± 32.0 mg/L). This is the first report where mutagenesis is applied in Aspergillus to improve ECB production. Further, fractional factorial design and central composite design were adopted to optimize the culture medium for increasing ECB production by the mutant ULN-59. Results indicated that four culture media including peptone, K2HPO4, mannitol and l-ornithine had significant effects on ECB production. The optimized medium provided another 1.4-fold increase in final ECB concentration to 2285.6 ± 35.6 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively ECB production from A. nidulans and could be a promising tool to improve other secondary metabolites production by fungal strains.
Co-reporter:Zhi-Qiang Liu;Wei Zheng;Jian-Feng Huang
Journal of Industrial Microbiology & Biotechnology 2015 Volume 42( Issue 8) pp:1091-1103
Publication Date(Web):2015 August
DOI:10.1007/s10295-015-1639-0
High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of kcat/Km was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.
Co-reporter:Zhi-Qiang Liu;Jing-Jing Ye;Zhen-Yang Shen
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 5) pp:2119-2129
Publication Date(Web):2015 March
DOI:10.1007/s00253-014-6245-y
(S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate to synthesize the side chain of cholesterol-lowering drug atorvastatin. To biosynthesize the (S)-CHBE, a recombinant Escherichia coli harboring the carbonyl reductase and glucose dehydrogenase was successfully constructed. The recombinant E. coli was cultured in a 500-L fermentor; after induction and expression, the enzyme activity and cell biomass were increased to 23,661.65 U/L and 13.90 g DCW/L which was 3.24 and 2.60-folds compared with those in the 50 L fermentor. The biocatalytic process for the synthesis of (S)-CHBE in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. The ethyl 4-chloro-3-oxobutanoate concentration reached to 1.7 M, and the (S)-CHBE with yield of 97.2 % and enantiomeric excess (e.e.) of 99 % was obtained after 4-h reaction in a 50-L reactor. In this study, the space-time yield and space-time yield per gram of biomass (dry cell weight, DCW) were 413.17 mM/h and 27.55 mM/h/g DCW for (S)-CHBE production, respectively, which were the highest values as compared to previous reports. Finally, (S)-CHBE was extracted from the reaction mixture with 82 % of yield and 95 % of purity. This study paved the foundation for the upscale production of (S)-CHBE by biocatalysis method.
Co-reporter:Dr. Nan-Wei Wan; Zhi-Qiang Liu;Dr. Feng Xue;Zhen-Yang Shen ; Yu-Guo Zheng
ChemCatChem 2015 Volume 7( Issue 16) pp:2446-2450
Publication Date(Web):
DOI:10.1002/cctc.201500453
Abstract
(S)-4-Chloro-3-hydroxybutyronitrile [(S)-CHBN] was used as a chiral building block for the preparation of atorvastatin. In this study, (R,S)-epichlorohydrin [(R,S)-ECH] and 1,3-dichloro-2-propanol (1,3-DCP) were investigated to prepare (S)-CHBN by using the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1. Preparing (S)-CHBN from (R,S)-ECH gave a modest enantiomeric excess (ee), whereas by using 1,3-DCP as the substrate, (S)-CHBN was obtained with 97.3 % ee after pH optimization. However, a low ee value and low yield of (S)-CHBN were obtained if the substrate concentration was increased to 10 g L−1. To obtain a higher ee value and yield, 16 mutants were constructed and screened. The variant W249F with improvements in activity and enantioselectivity was identified and applied at a 1,3-DCP loading of 10 g L−1, which gave (S)-CHBN in 86 % yield with 97.5 % ee in 1 h. This is the first report of a one-step biocatalytic process for the preparation of (S)-CHBN from prochiral 1,3-DCP.
Co-reporter:Ya-Ping Xue, Yu-Guo Zheng, Zhi-Qiang Liu, Xue Liu, Jian-Feng Huang, and Yin-Chu Shen
ACS Catalysis 2014 Volume 4(Issue 9) pp:3051
Publication Date(Web):July 30, 2014
DOI:10.1021/cs500535d
Enantiopure non-natural 2-aryl-amino acids are important intermediates for synthesizing pharmaceuticals. To develop an efficient chemoenzymatic process to produce non-natural 2-aryl-amino acids, a penicillin G acylase (PGA) gene from Bacillus megaterium was cloned and expressed in Bacillus subtilis WB800. The recombinant PGA exhibited a high hydrolytic activity and excellent enantioselectivity (E > 200) toward N-phenylacetyl derivatives of non-natural 2-aryl-amino acids. The l-2-aryl-amino acids were obtained in >99.9% enantiomeric excess (ee) and >49% conversion within 3 h. The position and type of the substituent in the substrate influence the recombinant PGA activity but do not affect the PGA enantioselectivity. The kinetic parameters of the recombinant PGA for different substrates were determined and compared. The mechanisms of enantioselectivity of PGA with respect to different substrates were elucidated. The chiral discrimination of PGA with respect to rac-2a–e was mainly because the d-substrates used in this study cannot interact with the active residues and bind to the active pocket as stably as the l-substrates. The unreacted N-phenylacetyl-d-2-aryl-amino acids can be completely racemized at 170 °C and then used as the substrate. A gram-scale production of l-2-aryl-amino acids was successfully achieved with approximately theoretical conversion, indicating that the chemoenzymatic approach appears to be promising for industrial applications.Keywords: amino acids; chiral resolution; deracemization; enzyme catalysis; hydrolysis; penicillin G acylase
Co-reporter:Nan-Wei Wan, Zhi-Qiang Liu, Kai Huang, Zhen-Yang Shen, Feng Xue, Yu-Guo Zheng and Yin-Chu Shen
RSC Advances 2014 vol. 4(Issue 109) pp:64027-64031
Publication Date(Web):19 Nov 2014
DOI:10.1039/C4RA13646B
We identified and characterized a novel halohydrin dehalogenase HHDH-PL from Parvibaculum lavamentivorans DS-1. Study of substrate specificity indicated that HHDH-PL possessed a high activity toward ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). After optimizations of the pH and temperature, whole cell catalysis of HHDH-PL was applied to the synthesis of ethyl (R)-4-cyano-3-hydroxybutyrate (HN) at 200 g L−1 of (S)-CHBE, which gave 95% conversion and 85% yield in 14 h.
Co-reporter:Zhi-Qiang Liu, Xin-Hong Zhang, Ya-Ping Xue, Ming Xu, and Yu-Guo Zheng
Journal of Agricultural and Food Chemistry 2014 Volume 62(Issue 20) pp:4685-4694
Publication Date(Web):April 28, 2014
DOI:10.1021/jf405683f
Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and kcat of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg–1 min–1, 24.45 s–1, and 9.24 mM, 47.68 μmol mg–1 min–1, and 34.55 s–1, respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(−)-mandelic acid.
Co-reporter:Xiao-Jun Li;Ren-Chao Zheng;Hong-Ye Ma
Applied Microbiology and Biotechnology 2014 Volume 98( Issue 6) pp:2473-2483
Publication Date(Web):2014 March
DOI:10.1007/s00253-013-5136-y
Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.
Co-reporter:Zhi-Qiang Liu;Ai-Cun Gao;Ya-Jun Wang
Journal of Industrial Microbiology & Biotechnology 2014 Volume 41( Issue 7) pp:1145-1158
Publication Date(Web):2014 July
DOI:10.1007/s10295-014-1443-2
A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of kcat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH.
Co-reporter:Zhong-Yu You;Zhi-Qiang Liu
Applied Microbiology and Biotechnology 2014 Volume 98( Issue 1) pp:11-21
Publication Date(Web):2014 January
DOI:10.1007/s00253-013-5357-0
Ethyl (R)-4-cyano-3-hydroxybutanoate (HN) is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin (Lipitor), which is the hydroxymethylglutaryl CoA reductase inhibitor. HN is also used as a synthon in the production of l-carnitine and (R)-4-amino-3-hydroxybutanoic acid. It is necessary to have a clear understanding of the synthesis process of HN for its extensive use. This review gives an overview of different synthetic strategies of optically active HN, including chemical and enzymatic approaches. The emphasis is focused mainly on the synthetic routes using biocatalysts, such as halohydrin dehalogenase, nitrilase, carbonyl reductase, and lipase.
Co-reporter:Li-Qun Jin;Zong-Tong Li;Zhi-Qiang Liu
Journal of Industrial Microbiology & Biotechnology 2014 Volume 41( Issue 10) pp:1479-1486
Publication Date(Web):2014 October
DOI:10.1007/s10295-014-1490-8
Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.
Co-reporter:Jin-Feng Zhang;Zhi-Qiang Liu;Xin-Hong Zhang
Chemical Papers 2014 Volume 68( Issue 1) pp:53-64
Publication Date(Web):2014 January
DOI:10.2478/s11696-013-0423-8
Biotransformation of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) was investigated with a newly isolated Alcaligenes faecalis ZJUTBX11 strain showing nitrilase activity in the immobilized form. To reduce the mass transfer resistance and to increase the toleration ability of the microorganisms to the toxic substrate as well as to enhance their ability to be reused, encapsulation of the whole cells in alginate-chitosan-alginate (ACA) membrane liquid-core capsules was attempted in the present study. The optimal pH and temperature for nitrilase activity of encapsulated A. faecalis ZJUTBX11 cells were 7.5°C and 35°C, respectively, which is consistent with free cells. Based on the Michaelis-Menten model, kinetic parameters of the conversion reaction with IDAN as the substrate were: Km = (17.6 ± 0.3) mmol L−1 and Vmax = (97.6 ± 1.2) μmol min−1 g−1 of dry cell mass for encapsulated cells and (16.8 ± 0.4) mmol L−1 and (108.0 ± 2.7) μmol min−1 g−1 of dry cell mass for free cells, respectively. After being recycled ten times, the whole cells encapsulated in ACA capsules still retained 90 % of the initial nitrilase activity while only 35 % were retained by free cells. Lab scale production of IDA using encapsulated cells in a bubble column reactor and a packed bed reactor were performed respectively.
Co-reporter:Zhong-Yu You;Zhi-Qiang Liu
Applied Microbiology and Biotechnology 2014 Volume 98( Issue 4) pp:1671-1680
Publication Date(Web):2014 February
DOI:10.1007/s00253-013-5042-3
A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg−1. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent Km and Vmax values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min−1 mg−1, respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous–organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP+ to (S)-CHBE were 26.5 mmol L−1 h−1 g−1 DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.
Co-reporter:Ya-Ping Xue, Yu-Guo Zheng, Ya-Qin Zhang, Jing-Lei Sun, Zhi-Qiang Liu and Yin-Chu Shen
Chemical Communications 2013 vol. 49(Issue 91) pp:10706-10708
Publication Date(Web):24 Sep 2013
DOI:10.1039/C3CC46240D
A facile and efficient one-pot, single-step method for deracemizing a broad range of 2-hydroxyacids to (R)-2-hydroxyacids was established by combination of resting cells of an (S)-hydroxyacid dehydrogenase-producing microorganism and an (R)-ketoacid reductase-producing microorganism.
Co-reporter:Ya-Ping Xue, Ming Xu, Hong-Sheng Chen, Zhi-Qiang Liu, Ya-Jun Wang, and Yu-Guo Zheng
Organic Process Research & Development 2013 Volume 17(Issue 2) pp:213-220
Publication Date(Web):January 15, 2013
DOI:10.1021/op3001993
An integrated bioprocess for the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid (R-MA) with immobilized Alcaligenes faecalis ZJUTB10 cells was constructed. Production of A. faecalis ZJUTB10 nitrilase in a pilot-scale fermenter (700 L) with high activity was achieved after optimizing cultivation conditions. A. faecalis ZJUTB10 cells were then immobilized in Ca-alginate. Efficient reusability of the biocatalyst up to 9 batches was obtained by immobilization, and treatment with polyethyleneimine (PEI) and glutaraldehyde (GA) further extended the longevity to 19 batches. The immobilized cells showed maximum activity at 40 °C and pH 8.0. A method for in situ product recovery (ISPR) based on an external extraction loop was established to overcome product inhibition. Anion-exchange column containing resin HZ202 was coupled to the packed bed bioreactor and enabled product recovery by continuously recirculating reaction mixture through the ISPR unit. This integrated bioprocess led to a high productivity of 8.87 mM/h after 16 h of reaction. The productivity of R-MA did not drop significantly even after 80 h of reaction, and the accumulative R-MA amount reached a final value of 550 mmol with excellent enantiomeric excess (>99%). The present studies demonstrated the potential of using the integrated bioprocess for continuous production of R-MA on an industrial scale.
Co-reporter:Zhong-Yu You;Zhi-Qiang Liu
Journal of Industrial Microbiology & Biotechnology 2013 Volume 40( Issue 1) pp:29-39
Publication Date(Web):2013 January
DOI:10.1007/s10295-012-1213-y
A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni2+, Ba2+ and Fe2+. The apparent Km and Vmax values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min−1 mg−1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min−1 mg−1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.
Co-reporter:Li-Qun Jin;Zhi-Qiang Liu;Jian-Miao Xu
World Journal of Microbiology and Biotechnology 2013 Volume 29( Issue 3) pp:431-440
Publication Date(Web):2013 March
DOI:10.1007/s11274-012-1195-y
In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.
Co-reporter:Ren-Chao Zheng;Tian-Zhen Wang;De-Jin Fu
Applied Microbiology and Biotechnology 2013 Volume 97( Issue 11) pp:4839-4847
Publication Date(Web):2013 June
DOI:10.1007/s00253-013-4810-4
The chemoenzymatic process involving biocatalytic resolution of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE, 1) has been the most competitive and attractive route for pregabalin. A new esterase-producing strain ZJB-09203, which exhibited high hydrolytic activity, excellent enantioselectivity, and diastereoselectivity towards CNDE, has been successfully isolated from soil samples with a pH indicator agar plate method. The isolate was identified as Morgarella morganii by the ATB system (ID 32 GN) and the 16S rDNA sequence. In order to suppress product inhibition during enzymatic hydrolysis of CNDE, an adsorptive biocatalytic process was developed by utilizing anion-exchange resin D201 as adsorbent for selective removal of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (2) from the reaction medium. This approach allowed the substrate loading to be increased up to 1.5 M and the chiral intermediate 2 was produced in 682 mM, 45.3 % conversion, and 95 % ee. These results imply that M. morganii ZJB-09203 esterase is a promising biocatalyst in the development of chemenzymatic manufacturing process for pregabalin.
Co-reporter:Zhong-Yu You;Zhi-Qiang Liu
Applied Microbiology and Biotechnology 2013 Volume 97( Issue 1) pp:9-21
Publication Date(Web):2013 January
DOI:10.1007/s00253-012-4523-0
Halohydrin dehalogenases (HHDHs) are lyases that catalyze the cleavage of carbon–halogen bond of halohydrins. They also can catalyze the reverse reaction in the presence of nucleophiles such as cyanide, azide, and nitrite ions. HHDHs have been recognized as the ideal tools for the degradation of various halogenated environmental pollutants. Moreover, they can be used as biocatalysts for the kinetic resolution of halohydrins and epoxides, and for the preparation of various substituted alcohols. This review is mainly focused on the current status of research on HHDHs, highlighting the production, characterization, structures and mechanism, protein engineering, and biotechnological applications of HHDHs.
Co-reporter:Jian-Miao Xu;Ben Chen;Yuan-Shan Wang
Chemical Papers 2013 Volume 67( Issue 10) pp:1262-1270
Publication Date(Web):2013 October
DOI:10.2478/s11696-013-0389-6
Bacterial strain ZJB-09211 capable of amidase production has recently been isolated from soil samples. The strain is able to asymmetrically hydrolyze l-tryptophanamide from d,l-tryptophanamide to produce l-tryptophan in high yield and with excellent stereoselectivity (enantiomeric excess > 99.9 %, and enantiomeric ratio > 200). Strain ZJB-09211 has been identified as Flavobacterium aquatile based on the cell morphology analysis, physiological tests, and the 16S rDNA sequence analysis. Optimization of the fermentation medium led to an about six-fold increase in the amidase activity of strain ZJB-09211, which reached 501.5 U L−1. Substrate specifity and stereoselectivity investigations revealed that amidase of F. aquatile possessed a broad substrate spectrum and high enantioselectivity.
Co-reporter:Shu-Ping Zou;Er-Hong Du;Zhong-Ce Hu
Biotechnology Letters 2013 Volume 35( Issue 6) pp:937-942
Publication Date(Web):2013 June
DOI:10.1007/s10529-013-1165-y
Biotransformation of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) by the whole cells of recombinant Escherichia coli expressing halohydrin dehalogenase was limited by product inhibition. To solve this problem and improve the ECH yield, a biotransformation strategy using resin-based in situ product removal (ISPR) was investigated. Seven macroporous resins were examined to adsorb ECH: resin HZD-9 was the best. When 10 % (w/v) HZD-9 was added to batch biotransformation, 53.3 mM ECH was obtained with a molar yield of 88.3 %. The supplement of the HZD-9 increased the ECH volumetric productivity from 0.5 to 2.8 mmol/l min compared to without addition of resin. In fed-batch biotransformation, this approach increased ECH from 31 to 87 mM. These results provide a promising basis for the biosynthesis of ECH.
Co-reporter:Zhi-Qiang Liu, Ling Zhang, Li-Hui Sun, Xiao-Jun Li, Nan-Wei Wan, Yu-Guo Zheng
Food Chemistry 2012 Volume 134(Issue 2) pp:948-956
Publication Date(Web):15 September 2012
DOI:10.1016/j.foodchem.2012.02.213
5′-Nucleotides including 5′-inosinic acid have characteristic taste and important application in various foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase) can catalyse the synthesis of 5′-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase was purified using nickel–NTA. The optimal temperature and pH of this enzyme were 30 °C and 5.0, respectively. The activity was partially inhibited by metal ions such as Hg2+, Ag+ and Cu2+, but not by chelating reagents such as EDTA. The values of Km and Vmax for inosine were 40 mM and 3.5 U/mg, respectively. Using this purified enzyme, 16.83 mM of 5′-IMP was synthesised from 37 mM of inosine and the molar yield reached 45.5%. Homology modelling and docking simulation were discussed.Highlights► Escherichia blattae JCM 1650 with mutant AP/PTase gene was synthesised and expressed in Escherichia coli. ► 5′-Inosine monophosphate (5′-IMP) was isolated and characterised. ► The molar yield of 5′-IMP reached 45.5%. ► This recombinant AP/PTase has potential for production of 5′-nucleotides.
Co-reporter:Huo-Xi Jin;Zhong-Ce Hu
Journal of Biosciences 2012 Volume 37( Issue 4) pp:695-702
Publication Date(Web):2012 September
DOI:10.1007/s12038-012-9243-1
The enantioselective hydrolysis of racemic epichlorohydrin for the production of enantiopure (S)-epichlorohydrin using whole cells of Aspergillus niger ZJB-09173 in organic solvents was investigated. Cyclohexane was used as the reaction medium based on the excellent enantioselectivity of epoxide hydrolase from A. niger ZJB-09173 in cyclohexane. However, cyclohexane had a negative effect on the stability of epoxide hydrolase from A. niger ZJB-09173. In the cyclohexane medium, substrate inhibition, rather than product inhibition of catalysis, was observed in the hydrolysis of racemic epichlorohydrin using A. niger ZJB-09173. The racemic epichlorohydrin concentration was markedly increased by continuous feeding of substrate without significant decline of the yield. Ultimately, 18.5% of (S)-epichlorohydrin with 98% enantiomeric excess from 153.6 mM of racemic epichlorohydrin was obtained by the dry cells of A. niger ZJB-09173, which was the highest substrate concentration in the production of enantiopure (S)-epichlorohydrin by epoxide hydrolases using an organic solvent medium among the known reports.
Co-reporter:Ren-Chao Zheng;Zheng Ge;Zhao-Kuan Qiu
Applied Microbiology and Biotechnology 2012 Volume 94( Issue 4) pp:969-976
Publication Date(Web):2012 May
DOI:10.1007/s00253-012-3942-2
Biocatalytic asymmetric preparation of (R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent (R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. (R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave (R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of (R)-1,3-butanediol.
Co-reporter:Ya-Ping Xue;Wei Wang;Ya-Jun Wang;Zhi-Qiang Liu
Bioprocess and Biosystems Engineering 2012 Volume 35( Issue 9) pp:1515-1522
Publication Date(Web):2012 November
DOI:10.1007/s00449-012-0741-1
To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (Eapp). The Eapp closely matches the value of true enantioselectivity (Etrue) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.
Co-reporter:Zhi-Jian Lin;Ren-Chao Zheng;Ya-Jun Wang
Journal of Industrial Microbiology & Biotechnology 2012 Volume 39( Issue 1) pp:133-141
Publication Date(Web):2012 January
DOI:10.1007/s10295-011-1008-6
A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l−1) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l−1, 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.
Co-reporter:Ya-Jun Wang, Ye-Fei Wu, Feng Xue, Zhi-Xian Wu, Ya-Ping Xue, Yu-Guo Zheng, Yin-Chu Shen
Journal of Chromatography B 2012 Volumes 895–896() pp:146-153
Publication Date(Web):1 May 2012
DOI:10.1016/j.jchromb.2012.03.033
Brefeldin A (BFA) is a macrolide lactone antibiotic, possessing antitumor, antiviral, antifungal activities. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography combined with crystallization was established for BFA purification from Eupenicillium brefeldianum CCTCC M 208113 fermentation broth. Among six macroporous resin adsorbents tested, the non-polar resin HZ830 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Freundlich equation, and the adsorption kinetic followed the pseudo-second order model. Through experimental optimization of column adsorption and desorption, BFA in purity of 90.4% (w/w), 92.1% (w/w) yield was obtained by a one-step macroporous resin adsorption chromatography, using a stepwise elution protocol. Furthermore, high purity (>99%, w/w) of BFA crystals were prepared from E. brefeldianum CCTCC M 208113 fermentation broth in an overall recovery of 67.0% (w/w), using a combination of adsorption chromatography packed with non-polar macroporous adsorbent HZ830 and crystallization in acetone.Highlights► One step isolation of brefeldin A from broth with macroporous resin is developed. ► The static equilibrium adsorption data of brefeldin A fits the Freundlich model. ► One-step column chromatography recovers 92.1% of brefeldin A in a purity of 90.4% from fermentation broth. ► A combination of adsorption chromatography and crystallization produces brefeldin A with purity of >99%.
Co-reporter:Ya-Jun Wang;Zhi-Qiang Liu;Ren-Chao Zheng
Journal of Industrial Microbiology & Biotechnology 2012 Volume 39( Issue 3) pp:409-417
Publication Date(Web):2012 March
DOI:10.1007/s10295-011-1029-1
In this work, a mild, efficient bioconversion of 2,2-dimethylcyclopropanecarbonitrile (DMCPCN) to 2,2-dimethylcyclopropanecarboxamide (DMCPCA) in distilled water system was developed. The isolate FW815 was screened using the enrichment culture technique, displaying strong DMCPCN hydratase activity, and was identified as Rhodococcus boritolerans based on morphological, physiological, biochemical tests and 16S rRNA gene sequencing. Cultivation outcomes indicated that R. boritolerans FW815 was a neutrophile, with a growth optimum of 28–32°C; its DMCPCN hydratase belonged to the Fe-type family, and was most active at 38–42°C, pH 7.0, with maximal activity of 4.51 × 104 U g−1 DCW. R. boritolerans FW815 was found to be DMCPCA amidase-negative, eliminating the contamination of dimethylcyclopropanecarboxylic acid. Moreover, it displayed high activity and acceptable reusability in the non-buffered distilled water system, comparable to those in pH 7.0 phosphate buffer (50.0 mmol l−1).
Co-reporter:Ren-Chao Zheng, Ai-Peng Li, Zhe-Ming Wu, Jian-Yong Zheng, Yu-Guo Zheng
Tetrahedron: Asymmetry 2012 Volume 23(22–23) pp:1517-1521
Publication Date(Web):15 December 2012
DOI:10.1016/j.tetasy.2012.10.011
(S)-3-Cyano-5-methylhexanoic acid ethyl ester is a valuable synthetic intermediate for pregabalin. Immobilized lipase PS from Pseudomonas cepacia was screened and shown to be the best biocatalyst for the enantioselective hydrolysis of 3-cyano-5-methylhexanoic acid ethyl ester, a racemic mixture involving a β-stereocenter. The optimum temperature and pH for the biocatalytic process were 35 °C and 6.0, respectively. Lipase PS IM exhibited a strong tolerance toward high substrate concentrations of up to 2.0 M (366 g/l). In the scaled-up biotransformation, (S)-3-cyano-5-methylhexanoic acid ethyl ester was produced in 0.89 M (162.9 g/l), 99.2% ee, and 44.5% yield. These results indicated that lipase PS IM catalyzed the preparation of (S)-3-cyano-5-methylhexanoic acid ethyl ester and could be used as an efficient route for the large-scale production of pregabalin.(S)-3-Cyano-5-methylhexanoic acid ethyl esterC10H17NO2[α]D20=-12.2 (c 1.1, CH3OH)Absolute configuration: (S)Source of chirality: Kinetic resolutionPotassium (S)-3-Cyano-5-methylhexanoateC8H12NO2K[α]D20=-22.5 (c 1.1, CH3OH)Absolute configuration: (S)Source of chirality: Kinetic resolution
Co-reporter:Ya-Ping Xue;Zhi-Qiang Liu;Ming Xu;Ya-Jun Wang
Journal of Chemical Technology and Biotechnology 2011 Volume 86( Issue 3) pp:391-397
Publication Date(Web):
DOI:10.1002/jctb.2528
Abstract
BACKGROUND: (R)-(-)-Mandelic acid (R-MA) is an important intermediate and chiral regent with broad uses. An efficient method for the separation of R-MA from the bioreaction mixture with high yield is of great importance, thus, the main objective of this work is to investigate the recovery of R-MA using an ion-exchange process.
RESULTS: The equilibrium isotherms for the separation of R-MA by resin HZ202 were obtained in the pH range 5.0–9.0 and temperature range 25–35 °C. The equilibrium data are well fitted by the Langmuir isotherm. Batch kinetic experiments showed that the mobility of R-MA− in solution was rapid and the R-MA−/OH− ion-exchange process reached equilibrium after about 60 min. Adsorption kinetics were analyzed by a linear driving force mass-transfer model, yielding good prediction of the kinetic behavior. In fixed bed column experiments, the breakthrough curves of R-MA from the solution on resin HZ202 were determined at different flow rates and R-MA was eluted with different concentrations of HCl. A favorable breakthrough curve and optimal eluant concentration were obtained. The results were used for the separation of R-MA biosynthesized from (R,S)-mandelonitrile with nitrilase, and separation was successfully achieved with above 90% recovery yield.
CONCLUSION: Resin HZ202 presents favorable behavior for the recovery of R-MA, in terms of capacity, kinetics, affinity, and susceptibility to regeneration. The results of this study provide an efficient method for R-MA recovery from bioreaction mixture and could potentially be used in industry. Copyright © 2010 Society of Chemical Industry
Co-reporter:Zhi-Qiang Liu, Li-Zhu Dong, Feng Cheng, Ya-Ping Xue, Yuan-Shan Wang, Jie-Nv Ding, Yu-Guo Zheng, and Yin-Chu Shen
Journal of Agricultural and Food Chemistry 2011 Volume 59(Issue 21) pp:11560-11570
Publication Date(Web):September 13, 2011
DOI:10.1021/jf202746a
Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7–8 and 40 °C, respectively. Mg2+ stimulated hydrolytic activity, whereas Cu2+, Co2+, Ni2+, Ag+, and Hg2+ showed a strong inhibitory effect. The Km and vmax for mandelonitrile were 4.74 mM and 15.85 μmol min–1 mg–1 protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(−)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.
Co-reporter:Ya-Ping Xue;Sai-Zhen Xu;Zhi-Qiang Liu
Journal of Industrial Microbiology & Biotechnology 2011 Volume 38( Issue 2) pp:337-345
Publication Date(Web):2011 February
DOI:10.1007/s10295-010-0778-6
(R)-(−)-Mandelic acid (R-MA) is an important intermediate with broad uses. Recently, R-MA production using nitrilase has been gaining more and more attention due to its higher productivity and enantioselectivity. In this work, a new bacterium WT10, which exhibited favorable nitrilase activity and excellent enantioselectivity for production of R-MA by enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile, was isolated and identified as a strain of Alcaligenes faecalis. In order to improve its nitrilase activity for industrial application, the wild-type strain WT10 was further subjected to mutagenesis using a combined LiCl–ultraviolet irradiation and low energy N+ ion beams implantation technique. A valuable mutant strain A. faecalis ZJUTB10 was obtained. The nitrilase specific activity of the mutant strain was greatly improved up to 350.8 U g−1, in comparison with wild-type strain WT10 of 53.09 U g−1. The reaction conditions for R-MA production by mutant strain A. faecalis ZJUTB10 were also optimized. Nitrilase activity in mutant strain showed a broad pH optimum at pH 7.7–8.5. The optimal temperature was 35°C. The highest production rate reached 9.3 mmol h−1 g−1. The results showed that mutant strain A. faecalis ZJUTB10 was a new candidate for efficient R-MA production from (R,S)-mandelonitrile and could potentially be used in industrial production.
Co-reporter:Zhi-Jian Lin;Ren-Chao Zheng;Yin-Chu Shen
Biotechnology Letters 2011 Volume 33( Issue 9) pp:1809-1813
Publication Date(Web):2011 September
DOI:10.1007/s10529-011-0623-7
Cells of a new isolate of Rhodococcusqingshengii harboring nitrile hydratase converted 2-amino-2,3-dimethylbutyronitrile into 2-amino-2,3-dimethylbutyramide (ADBA). Cells also hydrated a broad range of substrates including saturated, unsaturated and cyclic aliphatic nitriles. The microorganism tolerated KCN and ADBA up to 5 mM and 40 g/l, respectively. At 10°C, ADBA reached 33.8 g/l (yield 84.5%) which was 2.5 times that at 30°C.
Co-reporter:Zhong-Ce Hu
Applied Biochemistry and Biotechnology 2011 Volume 165( Issue 5-6) pp:1152-1160
Publication Date(Web):2011 November
DOI:10.1007/s12010-011-9332-x
The 1,3-dihydroxyacetone (DHA)-overproducing mutant of Gluconobacter oxydans was screened via UV mutagenesis to enhance the DHA production, and the DHA fermentation condition was optimized using the dissolved oxygen (DO) control strategy. The stable mutant G. oxydans ZJB11001 exhibits high DHA productivity and can tolerate high DHA concentrations. The optimal condition for DHA production by G. oxydans ZJB11001 in a 15-L fermentor required an initial medium containing 5 g/L yeast extract, 20 g/L glycerol, 0.5 g/L K2HPO4, 0.1 g/L MgSO4·7H2O. The glycerol feeding rate was manually controlled to maintain the glycerol concentration at 5–10 g/L range. The culture pH was maintained at 6.0 within the first 20 h, and then adjusted to 5.0 until the end of the fermentation. The DO concentration increased from 20% to 30% after 24 h of fermentation, and then to 40% after 60 h of fermentation. The maximum DHA concentration of 209.6 ± 6.8 g/L was achieved after 72 h of fed-batch fermentation at 30 °C.
Co-reporter:Zhi-Hua Feng;Yuan-Shan Wang
Biotechnology and Bioprocess Engineering 2011 Volume 16( Issue 5) pp:
Publication Date(Web):2011 October
DOI:10.1007/s12257-011-0033-7
Alpha-amylase inhibitors are widely used by the pharmaceutical and agricultural industries, such as the treatment of diabetes and obesity and insect controller. Here, we developed a colorimetric method to screen for α-amylase inhibitor producing strains or mutants with higher α-amylase inhibitor productivity. This method relies on absorbance changes at 402 nm that are due to the inhibition of α-amylase catalyzed hydrolysis of 2-Chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl-maltoside by α-amylase inhibitors. The assay can be performed on a microtiter plate, making it simple and convenient. Using this method, α-amylase inhibitor producing strains and mutants with higher α-amylase inhibitor productivity can be rapidly screened. One strain, ZJB-08196, with the highest α-amylase inhibition was isolated and identified as Actinoplanes utahensis, and one mutant with higher acarbose production was obtained by screening 3,000 variants using this method.
Co-reporter:Ya-Jun Wang;Li-Ling Liu;Zhi-Hua Feng
World Journal of Microbiology and Biotechnology 2011 Volume 27( Issue 12) pp:2759-2766
Publication Date(Web):2011 December
DOI:10.1007/s11274-011-0751-1
Acarbose is a clinically useful drug for the treatment of type II, insulin-independent diabetes as a hypoglycemic agent. An acarbose-overproducing strain ZJB-08196, indentified as Actinoplanes utahensis, was able to produce 4,210 mg l−1 of acarbose at 591 mOsm kg−1 with the optimized conditions at bench scale. Shake flask fermentation showed that maltose, glycerol and monosodium glutamate were supportive for acarbose production; soybean meal had higher bioavailability than corn steep liquor. Moreover, acarbose formation was not parallel with mycelial growth and the pattern of acarbose production by A. utahensis ZJB-08196 was the type of mixed-growth associated.
Co-reporter:Yuan-Shan Wang;Feng Cheng;Ren-Chao Zheng
World Journal of Microbiology and Biotechnology 2011 Volume 27( Issue 12) pp:2885-2892
Publication Date(Web):2011 December
DOI:10.1007/s11274-011-0769-4
Amidase is a promising synthesis tool for chiral amides and related derivatives. In the present study, the biochemical properties of the Delftiatsuruhatensis CCTCC M 205114 enantioselective amidase were determined for its potential application in chiral amides synthesis. D. tsuruhatensis CCTCC M 205114 amidase was purified 105.2 fold with total activity recovery of 4.26%. The enzyme is a monomer with a subunit of approximately 50 kDa by analytical gel filtration HPLC and SDS–PAGE. It had a broad substrate spectrum and displayed high enantioselectivity against R-2, 2-dimethylcyclopropane carboxamide and R-mandelic amide. The amidase was applied to enantioselective hydrolysis of R-2, 2-dimethylcyclopropane carboxamide from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate S-2, 2-dimethylcyclopropane carboxamide. This enzyme did not require metal ions for the hydrolysis reaction. Its optimal pH and temperature were 8.0 and 35°C, respectively. The Km and Vmax of the amidase for R-2, 2-dimethylcyclopropane carboxamide were 2.54 mM and 8.37 μmol min−1 mg protein−1, respectively. After 60 min of the reaction, R-2, 2-dimethylcyclopropane carboxamide was completely hydrolyzed, generating S-2, 2-dimethylcyclopropane carboxamide with a yield of 45.9% and an e.e. of above 99%. Therefore, this amidase can serve as a promising producer for S-2, 2-dimethylcyclopropane carboxamide and other amides.
Co-reporter:Li-Qun Jin;Zhi-Qiang Liu
World Journal of Microbiology and Biotechnology 2010 Volume 26( Issue 5) pp:817-823
Publication Date(Web):2010 May
DOI:10.1007/s11274-009-0238-5
Pyrazinamide has received considerable attention for its effective antibacterial action in the reappearance of tuberculosis and for its broad application in the chemical industry. In this study, a 2-cyanopyrazine-degrading bacterial strain, numbered ZJB-09104, was newly isolated and identified as Serratia marcescens, based on its physiological and biological tests, ATB system analysis, and 16S rDNA sequence analysis. The strain exhibits only nitrile hydratase (NHase) activity and this NHase belongs to the cobalt NHase family of enzymes. Thermostability tests suggested that the NHase is thermophilic with an optimum temperature of 50°C. The NHase was effective in converting nitriles to the corresponding amides under the conditions of temperature 50°C and time course 7 h, respectively.
Co-reporter:Hua-Ping Dong
Chromatographia 2010 Volume 71( Issue 1-2) pp:85-89
Publication Date(Web):2010 January
DOI:10.1365/s10337-009-1401-8
A convenient ion-pair LC procedure was firstly established for rapid analysis of ethyl 3-hydroxyglutarate (3-EHG) in an enzymatic-hydrolysis mixture, and the detection limit was as low as 0.45 μmol L−1; high repeatability was achieved with intra-day (n = 5) and inter-day (n = 5) relative standard deviation (RSD) values of 1.56 and 2.38%, respectively. The good linearity was established for 3-EHG concentration in the broad range from 0.005 to 0.30 mol L−1, with a coefficient (r) of 0.9992. (S)- and (R)-3-EHG were separated by normal-phase LC after simple derivatization with (R)-(+)-phenylethanamine, ee value (≥95%) of 3-EHG prepared by Lipase B catalyzed hydrolysis of diethyl 3-hydroxyglutatate (3-DHG) was determined after optimization of the mobile phase, and the RSD was 0.75% (n = 9) for repeatability. The results showed that the above methods were highly reproducible and reliable for analysis and separation of (S)-3-EHG from bioconversion mixture.
Co-reporter:Hua-Ping Dong;Zhi-Qiang Liu
Applied Microbiology and Biotechnology 2010 Volume 87( Issue 4) pp:1335-1345
Publication Date(Web):2010 July
DOI:10.1007/s00253-010-2584-5
(R)-ethyl-3-hydroxyglutarate with highly optical purity (≥99%) can be used as a novel precursor for synthesis of chiral side chain of rosuvastatin. In this study, a novel synthesis route of (R)-ethyl-3-hydroxyglutarate by whole microorganism cells from racemic ethyl 4-cyano-3-hydroxybutyate was created. A strain ZJB-0910 capable of transforming racemic β-hydroxy aliphatic nitrile was isolated by employing a screening method based on a colorimetric reaction of Co2+ ion with ammonia, and identified as Rhodococcus erythropolis based on its morphology, physiological tests, Biolog, and the 16S rDNA sequence. After cultivation in a sterilized medium with composition of 20 g glucose, 5 g yeast extract, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.2 g MgSO4·7H2O per liter at 30°C and 150 rpm for 48 h, the whole cells of R. erythropolis ZJB-0910 were prepared as a catalyst in (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate for synthesis of (R)-ethyl-3-hydroxyglutarate, without bearing hydrolase activity for the ester bond of ethyl 4-cyano-3-hydroxybutyate. Under the optimized biotransformation conditions of pH 7.5, 30°C, and 20 mM substrate concentration, (R)-ethyl-3-hydroxyglutarate with 46.2% yield (ee > 99%) was afforded, and its chemical structure was determined by ESI-MS, NMR, and IR. The apparent Michaelis constant Km and maximum rate Vmax for this biocatalytic reaction were 0.01 M and 85.6 μmol min−1 g−1, respectively.
Co-reporter:Jian-Fen Zhang;Yin-Chu Shen
Applied Biochemistry and Biotechnology 2010 Volume 162( Issue 4) pp:966-974
Publication Date(Web):2010 October
DOI:10.1007/s12010-009-8787-5
A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH 7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40 °C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca2+. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent Km value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.
Co-reporter:Zhong-Ce Hu;Yin-Chu Shen
Biotechnology and Bioprocess Engineering 2010 Volume 15( Issue 4) pp:651-656
Publication Date(Web):2010 August
DOI:10.1007/s12257-009-3068-2
This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.
Co-reporter:Yuan-Shan Wang;Ren-Chao Zheng;Jian-Miao Xu
Journal of Industrial Microbiology & Biotechnology 2010 Volume 37( Issue 5) pp:503-510
Publication Date(Web):2010 May
DOI:10.1007/s10295-010-0696-7
Immobilized cells of Delftia tsuruhatensis CCTCC M 205114 harboring R-amidase were applied in asymmetric hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide (R − 1) from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate (S)-2, 2-dimethylcyclopropane carboxamide (S − 1). Maximum R-amidase activity of 13.1 U/g wet cells (0.982 U/g beads) was obtained under conditions of 3% sodium alginate, 2.5% CaCl2, 15 h crosslinking and 2 mm bead size, which was 53.9% of that of free cells (24.3 U/g wet cells). In addition, characterization of the immobilized cells was examined. The optimum R − 1 hydrolysis conditions were identified as follows: substrate concentration 10 mM, pH 8.5, temperature 35°C and time course 40 min. Under optimum conditions, the maximum yield and enantiomeric excess of (R)-2, 2-dimethylcyclopropanecarboxylic acid were 49.5% and >99%, respectively. This afforded S − 1 with a yield >49% and an e.e. of 97.7%. With good operational stability and excellent enanotioselectivity, the immobilized cells could be potentially utilized in industrial production of S − 1.
Co-reporter:Mei Shen;Zhi-Qiang Liu
Biotechnology and Bioprocess Engineering 2009 Volume 14( Issue 6) pp:795-802
Publication Date(Web):2009 December
DOI:10.1007/s12257-008-0252-8
The medium components of nitrilase production by Arthrobacter nitroguajacolicus ZJUTB06-99 were optimized in this study. Effects of factors such as carbon sources, nitrogen sources, and inducers on nitrilase production were investigated. Glucose, yeast extract, and ε-caprolactam were chosen as the suitable components. Moreover, experiments were carried out to fix the concentration of three factors for the zero coded level of variables in the subsequent optimization. Response surface methodology (RSM) and central composite design (CCD) were employed for further optimization. A quadratic model was found to fit the nitrilase activity and the variables. The results revealed that the optimized medium contained (%, w/v) 2.80, glucose; 0.57, yeast extract; and 0.42, ε-caprolactam. Validation experiments were carried out under the optimized conditions and nitrilase activity of 107.49 U/L was close to the predicted activity 110.82 U/L. After optimization, the nitrilase activity attained 2.86 fold of activity compared to the unoptimized conditions and the conversion of acrylonitrile was significantly improved. The strain growth curve and nitrilase activity alteration in the course of culture were tested. The cells were suitably harvested after cultured for 72∼78 h.
Co-reporter:Yuguo Zheng, Xiaolong Chen and Yinchu Shen
Chemical Reviews 2008 Volume 108(Issue 12) pp:
Publication Date(Web):October 16, 2008
DOI:10.1021/cr068216s
Co-reporter:Zhiqiang Liu, Yin Li, Fengjie Cui, Lifeng Ping, Jiangning Song, Yaniv Ravee, Liqun Jin, Yaping Xue, Jianmiao Xu, Geng Li, Yajun Wang and Yuguo Zheng
Journal of Agricultural and Food Chemistry 2008 Volume 56(Issue 23) pp:11499-11506
Publication Date(Web):November 14, 2008
DOI:10.1021/jf802317q
The objective of this work is to investigate the effects of reaction conditions on the synthesis of octenyl succinic anhydride (OSA)-modified starch from waxy corn starch and to study the characteristics of the OSA-modified starch as well as its applications. A mathematical model was developed to investigate the influences of various processing condition factors on the production of the OSA-modified waxy corn starch production and predict the optimum reaction conditions. The maximal degree of substitution (DS) of OSA-modified waxy corn starch (0.0204) was predicted to occur when the starch concentration was 31.2%, the pH was 8.6, the reaction temperature was 33.6 °C, and the reaction time was 18.7 h. Repeated reactions for producing OSA-modified waxy corn starch were carried out in a 5 m3 reactor under the optimized conditions for verification of the model. The characteristics of modified waxy corn starch including infrared spectrum, scanning electron microscopy, and pasting property were tested and emulsification capacity of the OSA-modified starch were evaluated as well.
Co-reporter:Yu-Guo Zheng;Jing Chen;Zhi-Qiang Liu
Applied Microbiology and Biotechnology 2008 Volume 77( Issue 5) pp:
Publication Date(Web):2008 January
DOI:10.1007/s00253-007-1236-x
Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.
Co-reporter:Ren-Chao Zheng;Yin-Chu Shen
Biotechnology Letters 2007 Volume 29( Issue 7) pp:1087-1091
Publication Date(Web):2007 July
DOI:10.1007/s10529-007-9358-x
Kinetic resolutions play important roles in industrial biotransformations for production of optical pure compounds from racemic substrates. A simple method, based on enantiomeric excess of both substrate (eeS) and the corresponding product (eeP), was developed for determination of concentration of enantiomers in kinetic resolution. Since only relative quantity (ee) was required in the proposed method, calibration and cumbersome quantitative sample handling can be avoided and analytical accuracy can be greatly improved.
Co-reporter:Ren-Chao Zheng;Yin-Chu Shen
Applied Microbiology and Biotechnology 2007 Volume 74( Issue 1) pp:
Publication Date(Web):2007 February
DOI:10.1007/s00253-006-0642-9
A novel enantioselective amidase screening system was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron(III) complexes. The enantioselectivities of amidase were evaluated by employing (R, S)-2, 2-dimethyl cyclopropanecarboxamide (1), (S)-2, 2-dimethyl cyclopropanecarboxamide and their mixture as substrates concurrently under the same conditions. To prove the accuracy of the screening system, enantioselectivity of acyl transfer reaction (ET) and that of hydrolytic reaction (EH) was compared. With this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific avtivity for (R, S)-1.
Co-reporter:Jian-Fen Zhang;Zhi-Qiang Liu
Applied Microbiology and Biotechnology 2007 Volume 73( Issue 6) pp:1275-1281
Publication Date(Web):2007 January
DOI:10.1007/s00253-006-0619-8
3-Ketovalidoxylamine A C–N lyase is one of three key enzymes in the production of valienamine, which is a potent glucosidase inhibitor from validamycin A. N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C–N lyase, was prepared from N-p-nitrophenylvalidamine with free cells of Stenotrophomonasmaltrophilia CCTCC M 204024. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylenediaminetetraacetic acid. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was pH 6.0 and 30°C, respectively. N-p-Nitrophenyl-3-ketovalidamine was formed with a yield of 0.68 in the first batch.
Co-reporter:Ya-Jun Wang;Jian-Ping Xue
World Journal of Microbiology and Biotechnology 2007 Volume 23( Issue 3) pp:355-362
Publication Date(Web):2007 March
DOI:10.1007/s11274-006-9231-4
Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co2+-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.
Co-reporter:Zhiqiang Liu;Yin Li;Yingying Xu;Lifeng Ping
Applied Microbiology and Biotechnology 2007 Volume 74( Issue 1) pp:99-106
Publication Date(Web):2007 February
DOI:10.1007/s00253-006-0635-8
An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca2+ and Fe2+, while strongly inhibited by Ag+ and Hg+, and slightly inhibited by Cu2+, Zn2+, Ba2+, Ni+, EDTA–Na2 and fumarate.
Co-reporter:Xi Luo, Ya-Jun Wang, Yu-Guo Zheng
Enzyme and Microbial Technology (September 2015) Volume 77() pp:68-77
Publication Date(Web):1 September 2015
DOI:10.1016/j.enzmictec.2015.06.004
•Kluyveromyces lactis XP1461 was screened, harbouring the aldo-keto reductase activity.•KlAKR was cloned and expressed in E. coli and the enzyme was purified.•The KlAKR has broad substrate specificity to carbonyl compounds.•The KlAKR provided optically pure chiral alcohol for the majority of test substrates.An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (vmax), apparent Michaelis–Menten constant (Km) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (kcat) were calculated as 7.63 U mg−1, 0.204 mM, 4.42 mM and 697.4 min−1, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates.Download full-size image
Co-reporter:Ya-Jun Wang, Feng Xue, Ye-Fei Wu, Ya-Ping Xue, Yu-Guo Zheng
Journal of Bioscience and Bioengineering (September 2012) Volume 114(Issue 3) pp:262-267
Publication Date(Web):1 September 2012
DOI:10.1016/j.jbiosc.2012.04.006
In this work, a robust brefeldin A-synthesizing fungus, Eupenicillium brefeldianum ZJB082702, was bred from a Murraya paniculata endophytic fungus E. brefeldianum A1163. Using one-factor-at-a-time experimental design, optimization of media composition for E. brefeldianum ZJB082702 fermenting brefeldin A was conducted. Outcomes indicated that mixed carbon source and mixed nitrogen source were of c ritical importance to brefeldin A fermentation. After 6 d culture in the optimized fermentation media, composed of (g l−1) 13.33 starch, 26.67 glucose, 1.0 yeast extract powder, 1.0 corn steep liquor, 0.5 soybean meal, 0.75 NaNO3, 2.5 malt extract, 6.0 CaCO3, 3.0 MgSO4, 4.0 KH2PO4, 1.0 × 10−2 CuSO4, brefeldin A yield peaked at 1304.7 mg l−1, 648.2 mg l−1 in 500 ml baffled flask and 15 l stirred fermentor respectively, formed as a growth associated type of secondary metabolite based on fermentation profile analysis.Highlights► A brefeldin A overproducing fungus, E. brefeldianum ZJB082702, was bred. ► Mixed carbon, nitrogen sources were critical to brefeldin A synthesis. ► Brefeldin A is synthesized by E. brefeldianum in a growth associated manner. ► Six-day culture in baffled flasks gave a peak brefeldin A yield of 1304.7 mg l−1. ► Brefeldin A titer peaked at 648.2 mg l−1 in 15 l stirred fermentor.
Co-reporter:Hua-Ping Dong, Ya-Jun Wang, Yu-Guo Zheng
Journal of Molecular Catalysis B: Enzymatic (September 2010) Volume 66(Issues 1–2) pp:90-94
Publication Date(Web):1 September 2010
DOI:10.1016/j.molcatb.2010.03.009
Optically pure ethyl (S)-3-hydroxyglutarate [(S)-3-EHG] is used as a key precursor for synthesis of a variety of pharmaceutically important compounds. In this work, we established an efficient procedure for enantioselectively hydrolyzing diethyl 3-hydroxyglutarate (3-DHG) to optically active (S)-3-EHG employing immobilized Candida antarctica lipase B (Novozym 435). Under the optimized conditions: pH 7.0, agitation speed 200 rpm, temperature 40 °C, 3-DHG concentration 0.15 mol L−1, and enzyme loading 7 g L−1, (S)-3-EHG was prepared in above 95% ee value and 98.5% yield, and the reaction was free from external mass transfer and intra-particle diffusion limitations and kinetically controlled. The inhibitions of substrate (3-DHG) and product (3-EHG) were excluded because both displayed no decline in activity at elevated concentrations within the given ranges. In addition, ethanol, a byproduct of the reaction, inhibited lipase B following an uncompetitive inhibition pattern. The kinetic constants were obtained through non-linear regression, with values of Vmax 1.29 mmol min−1 g−1, Km 0.06 mol L−1, and Ki 0.37 mol L−1, respectively.
Co-reporter:Li-Qun Jin, Yu-Guo Zheng
Pesticide Biochemistry and Physiology (September 2009) Volume 95(Issue 1) pp:28-32
Publication Date(Web):1 September 2009
DOI:10.1016/j.pestbp.2009.05.001
Trehalase, with the target to control insects, nematodes and fungi, is of increasing interest and has been investigated extensively in recent years. Validamycin compounds, as competitive trehalase inhibitors and lead compounds with broad applications have attracted substantial attention as well. In this study, the characterizations of termites trehalase were investigated and the inhibitory effects of validamycin compounds on the termites trehalase were studied as well. Results showed that the termites trehalase is presumably belonging to the acid trehalase with optimal pH of 3.3 and optimal temperature of 37 °C. It was investigated that the concentrations of validoxylamine A (VAA), validoxylamine B (VBB), validamycin A (VA) and validamycin B (VB) required for 50% inhibition IC50 of termites trehalase were calculated to be 14.73 mg l−1, 20.80 mg l−1, 3.17 × 103 mg l−1and 2.24 × 103 mg l−1, respectively. The inhibition kinetic constant Ki values for the above validamycin compounds were 3.2 × 10−6 mol l−1, 1.03 × 10−5 mol l−1, 4.02 × 10−4 mol l−1and 2.69 × 10−4 mol l−1, respectively. Validoxylamine A appeared to be the most potential termites trehalase inhibitor among the four compounds.
Co-reporter:Li-Tao Ruan, Ren-Chao Zheng, Yu-Guo Zheng
Protein Expression and Purification (October 2016) Volume 126() pp:16-25
Publication Date(Web):1 October 2016
DOI:10.1016/j.pep.2016.05.006
•Two amidase signature (AS) enzymes were mined by an efficient genome mining approach.•The recombinant AS amidases showed low sequence similarity with other reported amidases.•BeAmi2 and BeAmi4 were purified to homogeneity and characterized in detail.•BeAmi4 displayed optimum activity at pH 8.5 and was stable in alkaline condition.•BeAmi2 was an effective catalyst for hydrolysis of nicotinamide and its derivatives.Amidases have received increasing attention for their significant potential in the production of valuable carboxylic acids. In this study, two amidases belonging to amidase signature family (BeAmi2 and BeAmi4) were identified and mined from genomic DNA of Brevibacterium epidermidis ZJB-07021 by an efficient strategy combining comparative analysis of genomes and identification of unknown region by high-efficiency thermal asymmetric interlaced PCR (HiTAIL-PCR). The deduced amino acid sequences of BeAmi2 and BeAmi4 showed low identity (< 40%) with other reported amidases. The two amidases displayed optimum activity toward a wide spectrum of substrates at a mild alkaline pH and 45 °C. Both of them were remarkably inactivated by serine-directed inhibitor and sulfhydryl-reducing agent. Kinetic analysis revealed that nicotinamide was the preferable substrate for both amidases and the chlorine substitutions on the pyridine ring had a negative effect on activity. The bioprocesses for hydrolysis of 100 mM nicotinamide, isonicotinamide, 2-chloronicotinamide and 5-chloronicotinamide with purified BeAmi2 (6 U mL−1) were complete in 60 min with full conversion except 2-chloronicotinamide. These results indicated BeAmi2 was an effective catalyst for hydrolysis of several nicotinamide derivatives.
Co-reporter:Shan Lin, Zhi-Qiang Liu, Ming Yi, Hui Wu, Feng Xu, Yu-Guo Zheng
Protein Expression and Purification (May 2017) Volume 133() pp:81-89
Publication Date(Web):1 May 2017
DOI:10.1016/j.pep.2017.03.005
•Two novel chitinases (chiA and chiH) were identified using transcriptome-mining.•The recombinant chiA and chiH were purified to homogeneity and characterized.•The thermostability and acid preference distinguish chiA and chiH from other reported family 18 chitinases.•chiA and chiH preferred to hydrolyze colloidal chitin and other substrates containing N-acetyl groups.Two novel family 18 chitinases, chiA and chiH, were identified and cloned from the transcriptome of H. sinensis based on the transcriptome sequence data. The recombinant chitinases were overexpressed in Escherichia coli BL21, subsequently purified and functionally characterized. The optimal temperature and pH for chiA were 55 °C and 5.0, respectively, and those for chiH were 50 °C and 5.0, respectively. The highest enzyme activities of 11.5 U/mg and 8.1 U/mg were obtained for chiA and chiH, respectively, when colloidal chitin was used as the substrate with Ba2+. chiA exhibited higher Vmax of 1.94 μmol/μg/h and kcat of 1.443 S−1 than those of chiH with Vmax of 1.63 μmol/μg/h and kcat of 1.175 S−1, and both were efficient towards colloidal chitin compared with other typical family 18 chitinases. Substrate specificity and gene expression analyses indicated that chiA and chiH preferred substrates containing N-acetyl groups, such as colloidal chitin and glycol chitin, while no activity was detected toward laminarin, cellobiose, carboxymethyl cellulose and starch. The work presented here would aid in the understanding and performance of future studies on the infection mechanism of H. sinensis.
Co-reporter:Feng Xue, Zhi-Qiang Liu, Ya-Jun Wang, Nan-Wei Wan, Yu-Guo Zheng
Journal of Molecular Catalysis B: Enzymatic (May 2015) Volume 115() pp:105-112
Publication Date(Web):1 May 2015
DOI:10.1016/j.molcatb.2015.02.008
•Cloning and expression of a novel HHDH from Tistrella mobilis ZJB1405.•HHDHTm exhibits higher activities toward halohydrins under more alkaline condition.•HHDHTm shows unusual enantioselectivities for the conversion of prochiral halohydrins.•The preparation of (S)-ECH from prochiral 1,3-DCP with more than 50% yield.•Biocatalysis of 100 g/L (S)-CHBE to (R)-HN with 89% recovered yield and >99% ee.A novel halohydrin dehalogenase (HHDHTm) has been identified in Tistrella mobilis ZJB1405 and subsequently cloned and over-expressed in Escherichia coli. HHDHTm shares low similarity (less than 38%), and has different biochemical catalytic characteristics from other HHDHs. The purified HHDHTm displays maximum enzymatic activity toward 1,3-dichloro-2-propanol (DCP) at the more alkaline pH, 45 °C. HHDHTm was stable up to 55 °C and approximately 60% of residual activity retained at 60 °C for 1 h. HHDHTm shows higher activities toward halohydrins with modest enantioselectivities compared with other reported HHDHs. Interesting, (S)-epichlorohydrin (ECH) was enantioselectively biotransformed from the prochiral 1,3-DCP with about 75% yield. Moreover, 100 g/L ethyl (S)-4-chloro-3-hydroxybutyrate (CHBE) can be converted to ethyl (R)-4-cyano-3-hydroxybutyrate (HN) with >99% ee and 89% isolated recovery by the recombinant HHDHTm. Therefore, HHDHTm is a promising biocatalyst for (S)-ECH and (R)-HN production.Download full-size image
Co-reporter:Zhong-Ce Hu, Sheng-Ying Tian, Li-Juan Ruan, Yu-Guo Zheng
Bioresource Technology (June 2017) Volume 233() pp:
Publication Date(Web):1 June 2017
DOI:10.1016/j.biortech.2017.02.096
•Achievement of near colorless DHA solution with few impurities.•Repeated production by continuous-feeding of glycerol and urea.•High DHA productivity and conversion rate of glycerol to DHA.•Immobilized cells on porous ceramics in a home-made bubble column bioreactor.Some inorganic nitrogen sources and amino acids instead of yeast extract, which resulted in trouble of product purification, were introduced for 1,3-dihydroxyacetone (DHA) production by biotransformation with Gluconobacter oxydans. The results showed that urea is an optimal nitrogen source. Furthermore, the effects of glycerol- and urea-feeding strategies for DHA production by immobilized cells in a home-made bubble column bioreactor were optimized. Cells immobilization was prepared by cultivation in the bioreactor packed with porous ceramics, and then the broth was removed. Then, repeated biotransformation by continuous-feeding of glycerol and urea was developed. Up to 96.4 ± 4.1 g/L of average DHA concentration with 94.8 ± 2.2% of average conversion rate of glycerol to DHA was achieved after 12 cycles of run. Near colorless DHA solution with few impurities was obtained and the production cost could be decreased.
Co-reporter:Yuan-Shan Wang, Xing-Chang Zheng, Qi-Wei Hu, Yu-Guo Zheng
Research in Microbiology (June 2015) Volume 166(Issue 5) pp:408-418
Publication Date(Web):1 June 2015
DOI:10.1016/j.resmic.2015.04.002
An abamectin (ABM)-degrading bacterium, Stenotrophomonas maltophilia ZJB-14120, was isolated and identified. This strain is capable of degrading 84.82% of ABM at an initial concentration of 200 mg/L over a 48 h incubation period. This strain showed efficient biodegradation ability (7.81 mg/L/h) to ABM and high tolerance (1000 mg/L) to all macrolides tested. In addition to ABM, emamectin, erythromycin and spiramycin can also be degraded by this strain. Modifications involving either reduction of the double bond between C22–C23 or replacement of the C25-group of ABM with a cyclohexyl group can completely inhibit biodegradation of ABM. The ABM-degrading capability of strain ZJB-14120 is likely to be intrinsic to its metabolism and could be inhibited by incubating with erythromycin, azithromycin, spiramycin or rifampicin. A new and successive degradation pathway was proposed based on metabolite analysis. Although there is evidence for metabolite inhibition, this strain has high ABM degradation activity and reusability. Further investigation showed that activated macrolide efflux pump(s) and an undetermined mechanism for regulating the intracellular ABM concentration are responsible for normal uptake of essential metabolites while pumping out excess harmful compounds. Strain ZJB-14120 may provide efficient treatment of water and soil contaminated by toxic levels of abamectin and emamectin.
Co-reporter:Zhe-Ming Wu, Ren-Chao Zheng, Yu-Guo Zheng
Protein Expression and Purification (January 2017) Volume 129() pp:60-68
Publication Date(Web):1 January 2017
DOI:10.1016/j.pep.2016.09.005
•A novel amidase signature (AS) amidase (Pl-Ami) was identified by genome mining.•The recombinant Pl-Ami were purified to homogeneity and characterized in detail.•The narrow substrate spectrum of Pl-Ami distinguish it from other AS amidases.•Pl-Ami preferred to hydrolyze long chain aliphatic amides.Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli BL21, purified and functionally characterized. The optimal pH and temperature for Pl-Ami were 9.5 and 45 °C, respectively. Pl-Ami preferred long chain aliphatic amides as substrates, while no activity was detected towards aromatic, heterocyclic and other amides. The highest enzyme activity of 128 U/mg was obtained when hexanoamide was used as substrate. Kinetic analysis indicated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover number (kcat) was higher with long chain aliphatic amides as substrates. The obtained results provided a distinct understanding of substrate specificity of AS family amidases.
Co-reporter:Jian-Fen Zhang, Yu-Guo Zheng, Yin-Chu Shen
Pesticide Biochemistry and Physiology (January 2007) Volume 87(Issue 1) pp:73-77
Publication Date(Web):1 January 2007
DOI:10.1016/j.pestbp.2006.06.001
Valienamine, an aminocyclitol with similar configuration to α-glucose, has a strong inhibitory effect on α-glucosidase. α-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase was investigated. Our results show that valienamine inhibition of honeybee α-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee α-glucosidase in vitro with an IC50 value of 5.22 × 10−5 M and Ki value of 3.54 × 10−4 M at pH 6.5, 45 °C. Valienamine has the potential to be developed into novel insecticides.
Co-reporter:Ya-Jun Wang, Xiao-Qing Liu, Xi Luo, Zhi-Qiang Liu, Yu-Guo Zheng
Journal of Molecular Catalysis B: Enzymatic (December 2015) Volume 122() pp:44-50
Publication Date(Web):1 December 2015
DOI:10.1016/j.molcatb.2015.08.018
A CaAKR was cloned from Candida albicans XP1463 and heterologously expressed in E. coli.The CaAKR has broad substrate specificity to various carbonyl compounds.It is able to synthesize some valuable optically pure chiral alcohol pharmaceutical intermediates.An aldo-keto reductase encoding gene caakr was cloned from Candida albicans XP1463 (CCTCC M 2014382), and heterologously expressed in Escherichia coli. The aldo-keto reductase CaAKR is NADH-dependent with a molecular weight of approximately 38.6 kDal including a His6-Tag. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (Vmax), apparent Michaelis⿿Menten constant (Km) and catalytic constant (kcat) for t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((R)-1a) were 11.50 mmol/L min, 1.91 mmol/L and 218.50 min⿿1. Besides atorvastatin's chiral synthon t-butyl 6-cyano-(3R,5R)- dihydroxy hexanoate ((R,R)-1b), it can synthesize N,N-2-dimethyl-(3S)-hydroxy-3-(2-thienyl)-1-propanine ((S)-9b) and methyl 1-[E]-2-[3-[3-[2-(7-chloro-2-quinoliny) ethenyl] phenyl]-(3S)-hydroxy propy] benzoate ((S)-10b), the chiral intermediates of duloxetine and montelukast, displaying potential applications in pharmaceutical industry.Download high-res image (166KB)Download full-size image
Co-reporter:Yuan-Shan Wang, Qi-Wei Hu, Xing-Chang Zheng, Jian-Fen Zhang, Yu-Guo Zheng
Journal of Microbiological Methods (April 2017) Volume 135() pp:93-95
Publication Date(Web):1 April 2017
DOI:10.1016/j.mimet.2017.02.007
•A screening method for oxo-AVM was developed based on its reaction with DNPH.•Combined with HPLC assay, regioselective AVM oxidizing microorganisms were obtained.•It is simple and rapid for the preliminary screening of AVM oxidizing biocatalysts.A spectrophotometric screening method for avermectin oxidizing microbes by determination of 4″-oxo-avermectin was established based on the reaction between 4″-oxo-avermectin and 2,4-dinitrophenylhydrazine. Combined with a gradient HPLC assay, microorganisms capable of regioselectively oxidizing avermectin to 4″-oxo-avermectin were successfully obtained by this method.
Co-reporter:Dong-Xu Jia, Lin Zhou, Yu-Guo Zheng
Enzyme and Microbial Technology (April 2017) Volume 99() pp:1-8
Publication Date(Web):1 April 2017
DOI:10.1016/j.enzmictec.2017.01.001
•Discovering novel thermostable T. oshimai GI via genome mining approach.•Enhancing its enzymatic activity and thermostability by adding Mn2+.•Higher catalytic efficiency of ToGI toward d-glucose owing to lower Km and higher kcat/Km.•Realization of preparation of HFCS at elevated temperature.Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95 °C and could retain more than 80% of initial activity in the presence of 20 mM Mn2+ at 85 °C for 48 h. The Km and kcat/Km values for ToGI were 81.46 mM and 21.77 min−1 mM−1, respectively. Furthermore, the maximum conversion yield of 400 g/L d-glucose to d-fructose at 85 °C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature.Download high-res image (170KB)Download full-size image
Co-reporter:Zhi-Qiang Liu, Si-Chuan Dong, Huan-Huan Yin, Ya-Ping Xue, Xiao-Ling Tang, Xiao-Jian Zhang, Jun-Yao He, Yu-Guo Zheng
Bioresource Technology (April 2017) Volume 229() pp:
Publication Date(Web):1 April 2017
DOI:10.1016/j.biortech.2016.12.098
•A newly synthesized SCR gene was expressed in E. coli for (S)-ET-5 production.•The bioproduction of (S)-ET-5 in a biphasic solvent was first constructed.•The (S)-ET-5 was achieved by bioconversion without extra addition of cofactor.•The conversion of 99.1%, d.e. > 99.9% was achieved at 150 g L−1 ET-4.(4S)-3-[(5S)-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing carbonyl reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5 L fermenter culture were 10.9 g DCW L−1 and 14900.3 U g−1 DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p-xylene as the optimal organic phase under optimized reaction conditions, and 150 g L−1 (4S)-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to (S)-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of (S)-ET-5.Download high-res image (263KB)Download full-size image
Co-reporter:Ren-Chao Zheng, Zhong-Yi Yang, Cong-Cheng Li, Yu-Guo Zheng, Yin-Chu Shen
Journal of Molecular Catalysis B: Enzymatic (April 2014) Volume 102() pp:161-166
Publication Date(Web):1 April 2014
DOI:10.1016/j.molcatb.2014.02.005
•Industrial bioprocess catalyzed by NHase and amidase was developed for (S)-1.•Activities of NHase and amidase in 1000-L fermentor reached 351,000 and 5880 U/L.•(S)-1 was recovered by a novel macroporous resin adsorption chromatography.•90.8 kg of (S)-1 were obtained with total yield of 38%.•The yield of (S)-1 increased to 53% after one recycling of by-product.An industrial one-pot, two-step bioprocess catalyzed by nitrile hydratase (NHase) and amidase was developed for (S)-2,2-dimethylcyclopropanecarboxamide (1), the key chiral intermediate of cilastatin. The key unit operations of the whole process including fermentative production of enzymes, biotransformation, isolation of product, and recycling of by-product were reported for the first time. The volumetric enzyme activities of NHase and amidase in 1000-L fermentor were enhanced to 351,000 and 5880 U/L, respectively. The two-step, one-pot biotransformation of rac-2,2-dimethylcyclopropanecarbonitrile (2) took full advantage of both enzymes, leading to accumulation of (S)-1 in 47% yield and 99.6% ee. (S)-1 and the by-product (R)-2,2-dimethylcyclopropanecarboxylic acid (3) were obtained with yields of 38% and 45%, respectively, by a novel macroporous resin adsorption chromatography. Moreover, the total yield of (S)-1 was further increased to 53% after one recycling of (R)-3. The new bioprocess dramatically improved process efficiency compared with the chemical route by elimination of six synthetic steps and proved to be a superior and more cost-effective approach towards (S)-1.Download full-size image
Co-reporter:Ya-Ping Xue, Ying-Peng Wang, Zhe Xu, Zhi-Qiang Liu, Xin-Rui Shu, Dong-Xu Jia, Yu-Guo Zheng, Yin-Chu Shen
Catalysis Communications (5 June 2015) Volume 66() pp:121-125
Publication Date(Web):5 June 2015
DOI:10.1016/j.catcom.2015.03.035
•An efficient chemoenzymatic route for the production of gabapentin was developed.•1-Cyanocyclohexaneacetic acid was synthesized by a greatly improved nitrilase.•A feasible procedure for producing the regioselective nitrilase was demonstrated.•Direct hydrogenation of 1-cyanocyclohexaneacetic acid was performed successfully.An efficient chemoenzymatic process is devised for synthesizing high-purity gabapentin. 1-Cyanocyclohexaneacetic acid was first produced in 0.94 M from 1.0 M 1-cyanocycloalkaneacetonitrile by a greatly improved nitrilase from Acidovorax facilis ZJB09122, resulting in a commercially attractive bioprocess with an outstanding space-time yield of 461 g/L/day. The resulting aqueous 1-cyanocycloalkaneacetic acid was then directly converted to 2-azaspiro [4.5] decan-3-one without further purification in subsequent hydrogenation by Raney-nickel, followed by simple chemical steps to afford gabapentin in high purity and 77.3% overall yield from 1-cyanocyclohexylacetonitrile. The simplicity of the process makes this new pathway suitable for large-scale preparation.Download full-size image
Co-reporter:Zhi-Qiang Liu, Li-Ping Zhang, Feng Cheng, Li-Tao Ruan, Zhong-Ce Hu, Yu-Guo Zheng, Yin-Chu Shen
Catalysis Communications (30 November 2011) Volume 16(Issue 1) pp:133-139
Publication Date(Web):30 November 2011
DOI:10.1016/j.catcom.2011.09.010
In the current study, an epoxide hydrolase (EH) gene from Rhodosporidium toruloides was synthesized and expressed in Escherichia coli. After purification, we found that the optimal pH and temperature of this enzyme were 7.5 and 35 °C, respectively. The recombinant EH obtained in this study was temperature-sensitive and the activity decreased significantly above 45 °C. The values of apparent Km and Vmax were 0.5953 mol/l and 0.0105 mol/(L·min). In addition, enantiomeric excesses value of (R)-epichlorohydrin could reach 100% after 40-min reaction. Moreover, this EH showed a broad substrates specificity toward epoxides. To the best of our knowledge, this is the first time to report the application of R. toruloides EH in racemic resolution of (R,S)-epichlorohydrin to produce (R)-epichlorohydrin.A newly synthesized epoxide hydrolase was obtained and characterized. This recombinant epoxide hydrolase could be used in racemic resolution of (R,S)-epichlorohydrin to produce (R)-epichlorohydrin.Download full-size imageHighlights► R. toruloides CBS14 EH gene was synthesized and expressed in E. coli. ► The recombinant EH showed a broad substrate specificity toward epoxides. ► The enantiomeric excess value of (R)-epichlorohydrin was 100%. ► This recombinant EH is potential for production of (R)-epichlorohydrin and diols.
Co-reporter:Shu-Ping Zou, Yu-Guo Zheng, Er-Hong Du, Zhong-Ce Hu
Journal of Biotechnology (20 October 2014) Volume 188() pp:42-47
Publication Date(Web):20 October 2014
DOI:10.1016/j.jbiotec.2014.08.014
•Use of biphasic system to improve (S)-2,3-dichloro-1-propanol productivity.•n-hexane was selected as the best organic phases.•Substrate concentration and biocatalyst productivity were significantly improved.•The efficient biosynthesis process was feasible on a 2-L preparative scale.The enantioselective resolution of (R,S)-2,3-dichloro-1-propanol ((R,S)-DCP) to (S)-DCP by whole cells of a recombinant Escherichia coli expressing halohydrin dehalogenase (HHDH) activity was limited by product inhibition. To solve this problem to improve the productivity of (S)-DCP, an n-heptane–aqueous biphasic system was adopted in this work. The influential operational parameters including phase volumetric ratio, buffer pH and reaction temperature were optimized. Under the optimal reaction conditions, significant improvements of substrate concentration and biocatalyst productivity (375 mM and 7.64 mmol (S)-DCP g−1 cell) were achieved in this n-heptane–aqueous biphasic system compared with aqueous single-phase system (150 mM and 2.97 mmol g−1 cell). The scale-up biosynthesis of (S)-DCP was successfully performed in a 2-L stirred reactor, resulting in a 128.8 mM (S)-DCP with enantiomeric excess of 99.1% and average productivity of 2.07 g (S)-DCP L−1 h−1, respectively.
Co-reporter:Xiao-Jun Li, Ren-Chao Zheng, Hong-Ye Ma, Jian-Feng Huang, Yu-Guo Zheng
Journal of Biotechnology (20 October 2014) Volume 188() pp:29-35
Publication Date(Web):20 October 2014
DOI:10.1016/j.jbiotec.2014.08.004
•Three complementary strategies were used to improve the activity of a lipase.•Several mutants with enhanced activity were obtained and characterized.•The key residue for enhancing catalytic efficiency of lipase was revealed.•The engineered lipase was used for the kinetic resolution of CNDE at a very high substrate loading.•New insights into the lipase structure–function relationship were provided.The variant Lip-T (S88T/A99N/V116D) of lipase Lip from Thermomyces lanuginosus has been proved to be a potential biocatalyst for kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) to produce valuable chiral intermediate of Pregabalin. In this study, random, site-directed and site-saturation mutagenesis were performed to further enhance the activity of Lip-T, and the key residues responsible for catalytic efficiency were revealed. A mutant S63L/D232A with improved activity toward CNDE was obtained after screening of approximately 2500 clones from random-mutant libraries. Site-directed mutagenesis at site 63 and 232 demonstrated that the single-point mutants S63L and D232A showed opposite effect on activity. S63L exhibited a significant improvement on activity, whereas D232A exerted a slight inhibitory effect. Then a mutant S63M with a 4.5-fold higher catalytic efficiency than Lip-T was obtained by site-saturation mutagenesis. Structural changes resulting from the mutations were analyzed and the mechanisms responsible for the enhanced activity were discussed. Moreover, the engineered lipase catalyzed enantioselective hydrolysis of CNDE at a very high substrate loading (765 g/l). As only 5% (w/v) resting cells were used, the bioprocess is much more cost-effective than Pfizer's process using 8% (w/v) commercially available lipase Lipolase®. These results provide not only new insights into lipase structure–function relationships but also a novel robust biocatalyst for the production of Pregabalin.
Co-reporter:Ji-Geng Hu, Ya-Jun Wang, Yu-Guo Zheng, Yin-Chu Shen
Enzyme and Microbial Technology (2 August 2007) Volume 41(Issue 3) pp:244-249
Publication Date(Web):2 August 2007
DOI:10.1016/j.enzmictec.2007.01.014
To isolate glycolonitrile-hydrolyzing microorganisms, a simple and rapid high-throughput screening method based on a colorimetric reaction of glycolic acid with β-naphthol in sulfuric acid solution was developed. Four strains able to convert glycolonitrile to glycolic acid were isolated from soil samples using this screening method, among which Rhodococcus sp. ZJUT-N595 displayed the highest hydrolytic activity. Resting cells of Rhodococcus sp. ZJUT-N595 could also biotransform acrylonitrile, racemic 2,2-dimethylcyclopropanecarbonitrile to corresponding amides as well as carboxylic acids, and racemic 2,2-dimethylcyclopropanecarboxamide to (S)-2,2-dimethylcyclopropanecarboxlic acid (e.e. > 90%), which demonstrated that this strain contained both intracellular nitrile hydratase and amidase activities. Batch cultivation showed that the optimal growth temperature and initial medium pH for Rhodococcus sp. ZJUT-N595 glycolonitrile-hydrolyzing activity were 30 °C and pH 8, respectively. The preferred carbon and nitrogen source and ideal inducer were mannitol, a composite of peptone plus yeast extract and caprolactam.
Co-reporter:Ya-Ping Xue, Fang-Fang Tian, Li-Tao Ruan, Zhi-Qiang Liu, Yu-Guo Zheng, Yin-Chu Shen
Journal of Biotechnology (10 September 2013) Volume 167(Issue 3) pp:271-278
Publication Date(Web):10 September 2013
DOI:10.1016/j.jbiotec.2013.06.015
•Concurrent obtaining of aromatic (R)-2-HA and aromatic 2-KA was demonstrated.•P. aeruginosa ZJB1125 displayed pretty high enantioselectivity for aromatic 2-HA.•An efficient method was established to separate aromatic (R)-2-HA and aromatic 2-KA.•A novel methodology was provided to utilize racemic aromatic 2-HA efficiently.Pseudomonas aeruginosa ZJB1125 harboring a stereoselective 2-hydroxyacid dehydrogenase (2-HADH) can catalyze asymmetric oxidation of mandelic acid and 2-chloromandelic acid into (R)-isomers and corresponding 2-ketoacids with high activity and enantioselectivity, while no consecutive oxidation of 2-ketoacids was observed during whole-cell catalysis. The 2-HADH in P. aeruginosa ZJB1125 is a FMN-dependent flavoprotein and did not require NAD(P)+ as cofactors to catalyze the oxidation reaction. Enzyme activity staining identified 2-HADH as the key enzyme that enantioselectively oxidized (S)-hydroxyacid to 2-ketoacid. The 2-HADH in P. aeruginosa ZJB1125 is inducible and 2-chloromandelic acid was found to induce its synthesis efficiently. The bacterium displayed pretty high activity and enantioselectivity for most of the aromatic 2-hydroxyacids examined, and have a potential for the concurrent obtaining of aromatic (R)-2-hydroxyacids and aromatic 2-ketoacids in near theoretical conversions. Using a simple organic extract process, aromatic (R)-2-hydroxyacids and aromatic 2-ketoacids can be effectively separated from the biocatalytic reaction mixture with high yield (>95%). This work provided a novel method for the concurrent obtaining of aromatic (R)-2-hydroxyacids and aromatic 2-ketoacids by oxidation of aromatic 2-hydroxyacids in one-step biotransformation, which would be a valuable process due to its high atom economy.
Co-reporter:Xiang Chen, Zhi-Qiang Liu, Jian-Feng Huang, Chao-Ping Lin and Yu-Guo Zheng
Chemical Communications 2015 - vol. 51(Issue 61) pp:NaN12331-12331
Publication Date(Web):2015/06/22
DOI:10.1039/C5CC04652A
Three short-chain alcohol dehydrogenases from Burkholderia gladioli were discovered for their great potential in the dynamic kinetic asymmetric transformation of methyl 2-benzamido-methyl-3-oxobutanoate, and their screening against varied organic solvents and substrates. This is the first report of recombinant enzymes capable of achieving this reaction with the highest enantio- and diastereo-selectivity.
Co-reporter:Ya-Ping Xue, Yu-Guo Zheng, Ya-Qin Zhang, Jing-Lei Sun, Zhi-Qiang Liu and Yin-Chu Shen
Chemical Communications 2013 - vol. 49(Issue 91) pp:NaN10708-10708
Publication Date(Web):2013/09/24
DOI:10.1039/C3CC46240D
A facile and efficient one-pot, single-step method for deracemizing a broad range of 2-hydroxyacids to (R)-2-hydroxyacids was established by combination of resting cells of an (S)-hydroxyacid dehydrogenase-producing microorganism and an (R)-ketoacid reductase-producing microorganism.
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