Co-reporter:Xiao-Wei Yu, Min Yang, Chuanhuan Jiang, Xiaofeng Zhang, and Yan Xu
Journal of Agricultural and Food Chemistry July 26, 2017 Volume 65(Issue 29) pp:6009-6009
Publication Date(Web):July 6, 2017
DOI:10.1021/acs.jafc.7b01884
Our previous studies demonstrated that the N-glycans in Rhizopus chinensis lipase (RCL) was important for its secretion. In order to improve the secretion of Rhizopus oryzae lipase (ROL) under the control of the GAP promoter in Komagataella phaffii, two extra N-glycosylation sites were introduced in ROL according to the position of the N-glycosylation sites of RCL by sequence alignment. The results indicated that the secretion level of ROL was strongly improved by N-glycosylation engineering, and the highest value of extracellular enzyme activity was increased from 0.4 ± 0.2 U/mL to 207 ± 6 U/mL in a shake flask. In the 7-L fermenter, the extracellular enzyme activity of the mutant (2600 ± 43 U/mL) and the total protein concentration (2.5 ± 0.2 g/L) were 218- and 6.25-fold higher than these of the parent, respectively. This study presents a strategy for constitutive recombinant expression of ROL using the GAP promoter combined with N-glycosylation engineering, providing a potential enzyme for application in the food industry.Keywords: GAP promoter; Komagataella phaffii; lipase; N-glycosylation; rational design; Rhizopus oryzae;
Co-reporter:Qun Wu, Jianchun Lin, Kaixiang Cui, Rubin Du, Yang Zhu, and Yan Xu
Journal of Agricultural and Food Chemistry December 20, 2017 Volume 65(Issue 50) pp:11133-11133
Publication Date(Web):November 27, 2017
DOI:10.1021/acs.jafc.7b04099
Urea is the primary precursor of the carcinogen ethyl carbamate in fermented foods. Understanding urea metabolism is important for controlling ethyl carbamate production. Using Chinese liquor as a model system, we used metatranscriptome analysis to investigate urea metabolism in spontaneous food fermentation processes. Saccharomyces cerevisiae was dominant in gene transcription for urea biosynthesis and degradation. Lysinibacillus sphaericus was dominant for urea degradation. S. cerevisiae degraded 18% and L. sphaericus degraded 13% of urea in their corresponding single cultures, whereas they degraded 56% of urea in coculture after 12 h. Compared to single cultures, transcription of CAR1, DAL2, and argA, which are related to urea biosynthesis, decreased by 51, 36, and 69% in coculture, respectively. Transcription of DUR1 and ureA, which are related to urea degradation, increased by 227 and 70%, respectively. Thus, coexistence of the two strains promoted degradation of urea via transcriptional regulation of genes related to urea metabolism.Keywords: ethanol fermentation; ethyl carbamate; Lysinibacillus sphaericus; Saccharomyces cerevisiae; spontaneous food fermentation; urea;
Co-reporter:Jing Li, Rongzhen Zhang, Yan Xu, Rong Xiao, Kunpeng Li, Haiyan Liu, Jiawei Jiang, Xiaotian Zhou, Lihong Li, Lixian Zhou, Yang Gu
Process Biochemistry 2017 Volume 56(Volume 56) pp:
Publication Date(Web):1 May 2017
DOI:10.1016/j.procbio.2017.03.003
•We designed seven mutations to improve the xylose oxidation activity of BsGDH.•The xylose oxidation activities of A258F, A258H, A258W and A258Y were improved.•Kinetic analysis clarified the activity improvement of four mutants towards xylose.•A258F retained good organic solvent resistance, as with wild-type enzyme.Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg−1 and nearly 8-folds higher kcat/Km value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.Download high-res image (147KB)Download full-size image
Co-reporter:Xiao-Dong Wang;Ting Jiang;Xiao-Wei Yu
Journal of Industrial Microbiology & Biotechnology 2017 Volume 44( Issue 7) pp:1053-1063
Publication Date(Web):28 March 2017
DOI:10.1007/s10295-017-1938-8
Prolyl endopeptidase (PEP) is very useful in various industries, while the high cost of enzyme production remains a major obstacle for its industrial applications. Pichia pastoris has been used for the PEP production; however, the fermentation process has not be investigated and little is known about the impact of excessive PEP production on the host cell physiology. Here, we optimized the nitrogen source to improve the PEP expression level and further evaluated the cellular response including UPR and ERAD. During methanol induction phase the PEP activity (1583 U/L) was increased by 1.48-fold under the optimized nitrogen concentration of NH4+ (300 mmol/L) and casamino acids [1.0% (w/v)] in a 3-L bioreactor. Evaluated by RT-PCR the UPR and ERAD pathways were confirmed to be activated. Furthermore, a strong decrease of ERAD-related gene transcription was observed with the addition of nitrogen source, which contributed to a higher PEP expression level.
Co-reporter:Sha Sha, Shuang Chen, Michael Qian, Chengcheng Wang, and Yan Xu
Journal of Agricultural and Food Chemistry 2017 Volume 65(Issue 1) pp:
Publication Date(Web):December 17, 2016
DOI:10.1021/acs.jafc.6b04242
The aroma profile of Chinese roasted sesame-like flavor type liquor was investigated by means of headspace solid phase microextraction–aroma extract dilution analysis (HS-SPME-AEDA). Sixty-three odor-active regions were detected by HS-SPME-AEDA with flavor dilution (FD) factors >5, and 58 of these were further identified. Among them, ethyl hexanoate, 2-furfurylthiol, dimethyl trisulfide, 3-methylbutanal, ethyl butanoate, ethyl 2-methylbutanoate, ethyl pentanoate, and ethyl 4-methylpentanoate appeared with the highest FD factors. In particular, eight sulfur-containing odorants were identified to be potentially important to roasted sesame-like flavor type liquor. The concentration of these odor-active compounds was further quantitated by combination of four different quantitative measurements, and 36 odorants had concentrations higher than their corresponding odor thresholds. On the basis of the odor activity values (OAVs), 2-furfurylthiol (OAV 1182), dimethyl trisulfide (OAV 220), β-damascenone (OAV 116), and methional (OAV 99) could be responsible for the unique aroma of roasted sesame-like flavor type liquor. An aroma recombination model prepared by mixing 36 aroma compounds with OAVs >1 showed a good similarity to the aroma of the original roasted sesame-like flavor type liquor. For the first time, 2-furfurylthiol was determined to be a typical potent odorant in roasted sesame-like flavor type liquor by omission study.Keywords: 2-furfurylthiol; aroma recombinate; Chinese roasted sesame-like flavor type liquor; HS-SPME-AEDA; OAV;
Co-reporter:Yue MaKe Tang, Yan Xu, Ji-ming Li
Journal of Agricultural and Food Chemistry 2017 Volume 65(Issue 2) pp:
Publication Date(Web):December 25, 2016
DOI:10.1021/acs.jafc.6b04509
The key aroma compounds of Chinese Vidal icewine were characterized by means of gas chromatography–olfactometry (GC-O) coupled with mass spectrometry (MS) on polar and nonpolar columns, and their flavor dilution (FD) factors were determined by aroma extract dilution analysis (AEDA). A total of 59 odor-active aroma compounds in three ranks of Vidal icewines were identified, and 28 odorants (FD ≥ 9) were further quantitated for aroma reconstitution and omission tests. β-Damascenone showed the highest FD value of 2187 in all icewines. Methional and furaneol were first observed as important odorants in Vidal icewine. Aroma recombination experiments revealed a good similarity containing the 28 important aromas. Omission tests corroborated the significant contribution of β-damascenone and the entire group of esters. Besides, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol) and 3-(methylthio)-1-propanal (methional) also had significant effects on icewine character, especially on apricot, caramel, and tropical fruit characteristics.Keywords: aroma recombination and omission; gas chromatography−olfactometry (GC-O); odor active compounds; Vidal icewine;
Co-reporter:Hai Du, Bo Liu, Xueshan Wang, Yan Xu
International Journal of Food Microbiology 2017 Volume 260(Volume 260) pp:
Publication Date(Web):2 November 2017
DOI:10.1016/j.ijfoodmicro.2017.08.016
•During the liquor-making process, p-cresol was only detected in pit mud.•Prokaryotic communities in pit mud were revealed by 16 s rDNA MiSeq Sequencing.•p-cresol was mainly produced by the genus of Clostridium in pit mud.•p-cresol-producing Clostridium could be inhibited by Lactobacillus.The compound p-cresol is the major off-odor and toxic component of strong aroma-type Chinese liquor. To trace its origin, the p-cresol contents in the liquor-making process were detected by gas chromatography-mass spectrometry. The prokaryotic communities involved in the process were revealed by 16S rRNA gene MiSeq sequencing. The results showed that the microbial diversity and concentration of p-cresol in pit mud significantly increased with increased pit depth. Canonical correspondence analysis further revealed that p-cresol was positively correlated with the genera Aminobacterium, Clostridium, Sedimentibacter and Syntrophomonas. On investigating potential p-cresol producers, we obtained 11 species from pit mud samples using selective culture media. Clostridium butyricum, Clostridium tyrobutyricum, Clostridium aminovalericum and Eubacterium contortum were confirmed to possess the capacity for p-cresol production. Moreover, based on volatile compounds data, these strains were assigned to the same clusters characterized by the high abundance of butanoic acid and p-cresol. Furthermore, 24 pairs of significant correlations were identified from 14 genera using co-occurrence network analysis. Clostridium was the hub in pit mud and could be inhibited by increasing levels of Lactobacillus. These findings represented a step forward for controlling p-cresol in a complex microbial community of Chinese liquor.
Co-reporter:Qun Wu, Weian Zhu, Wei Wang, Yan Xu
Food Chemistry 2015 Volume 168() pp:390-395
Publication Date(Web):1 February 2015
DOI:10.1016/j.foodchem.2014.07.069
•Terpenoids formation was linked to a unique yeast community in liquor making.•Ten yeast species were detected in Chinese light style liquor fermentation.•S. cerevisiae, P. kudriavzevii, W. anomala produced various types of terpenoids.•Barley, pea and sorghum served diverse functions on terpenoids formation.Terpenoids are important trace flavour constituents in Chinese light-style liquors, and are formed by the various yeast species present during fermentation of liquor from cereal and legume materials. Saccharomyces cerevisiae, Pichia kudriavzevii and Wickerhamomyces anomalus are three such yeast species, and we found S. cerevisiae capable of generating thirteen different terpenoids in cereal and legume extract fermentation, by both de novo and biotransformation pathways. We also found that cereals such as sorghum and barley, and legumes such as peas, contained different terpenoids precursors, which differentially affected the formation and profile of terpenoids mixtures. This work gives new insights into the role of yeast species in generating the various terpenoids mixtures found in Chinese light-style liquors.
Co-reporter:Hai Du, Hu Lu, and Yan Xu
Journal of Agricultural and Food Chemistry 2015 Volume 63(Issue 1) pp:290-296
Publication Date(Web):December 9, 2014
DOI:10.1021/jf503351w
Diverse Streptomyces species act as geosmin producers in the Chinese liquor-making process, causing an earthy, off-odor containment. Through microbiological and metabolite analyses, this paper investigates the influence of several geosmin-producing Streptomyces on the microbial community of a brewing system. The antifungal activity against functional liquor-brewing microbes was assayed by an agar diffusion method. Several Streptomyces, most notably Streptomyces sampsonii QC-2, inhibited the growth of the brewing functional yeasts and molds in pure culture. In a simulated coculture, Streptomyces spp. reduced the flavor compounds (alcohols and esters) contributed by yeasts. Nine components in Streptomyces sampsonii QC-2 broth were detected by ultraperformance liquid chromatography coupled with photo diode array (UPLC–PDA), with characteristic ultraviolet absorptions at 360, 380, and 400 nm. The main products of Streptomyces sampsonii QC-2 were identified by ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF–MS/MS), and confirmed by standard mass spectrometry. The antifungal active components were revealed as a series of heptaene macrolide antibiotics.
Co-reporter:Qun Wu, Rong Zhang, Suqin Peng, and Yan Xu
Journal of Agricultural and Food Chemistry 2015 Volume 63(Issue 3) pp:888-893
Publication Date(Web):January 6, 2015
DOI:10.1021/jf5036806
This work investigated the biosynthetic mechanism of lichenysin, the newly identified nonvolatile matrix component in Chinese liquors. Transcriptomes were analyzed in three producers, Bacillus licheniformis CGMCC 3961, 3962, and 3963, which were isolated from Maotai-flavor liquor-making process and produced 386.3, 553.5, and 795.2 μg/L lichenysin in a simulative liquor fermentation process. Lichenysin synthetase genes lchAA–AD in these three producers were expressed much more highly than those of the nonproducer B. licheniformis ATCC 14580 (>18.4-fold). In addition, ABC transporters were the most significant responsive metabolic pathway, and the expression levels of peptide transporter genes dppABCDE all increased more than 19.2-fold. When B. licheniformis CGMCC 3963 was cultured in synthetic medium, the expression of dppABCDE and lichenysin both increased with the addition of casein hydrolysate (containing various peptides). This indicated that peptide would act as a substrate for lichenysin synthesis. This work sheds new light on the mechanism for lichenysin biosynthesis.
Co-reporter:Chao Kang;Xiao-Wei Yu
Journal of Industrial Microbiology & Biotechnology 2015 Volume 42( Issue 2) pp:263-272
Publication Date(Web):2015 February
DOI:10.1007/s10295-014-1571-8
A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca2+ and inhibited by Zn2+, Mn2+, Al3+, and Cu2+. The Km and kcat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.
Co-reporter:Guo Cui Mu;Yao Nie;Xiao Qing Mu
Applied Biochemistry and Biotechnology 2015 Volume 176( Issue 6) pp:1736-1745
Publication Date(Web):2015 July
DOI:10.1007/s12010-015-1675-2
Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of Vmax and kcat/Km, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently.
Co-reporter:Xing Meng;Qun Wu;Li Wang;Diqiang Wang
Journal of Industrial Microbiology & Biotechnology 2015 Volume 42( Issue 12) pp:1601-1608
Publication Date(Web):2015 December
DOI:10.1007/s10295-015-1647-0
Microbial interactions could impact the metabolic behavior of microbes involved in food fermentation, and therefore they are important for improving food quality. This study investigated the effect of Bacillus licheniformis, the dominant bacteria in the fermentation process of Chinese Maotai–flavor liquor, on the metabolic activity of Saccharomyces cerevisiae. Results indicated that S. cerevisiae inhibited the growth of B. licheniformis in all mixed culture systems and final viable cell count was lower than 20 cfu/mL. Although growth of S. cerevisiae was barely influenced by B. licheniformis, its metabolism was changed as initial inoculation ratio varied. The maximum ethanol productions were observed in S. cerevisiae and B. licheniformis at 106:107 and 106:108 ratios and have increased by 16.8 % compared with single culture of S. cerevisiae. According to flavor compounds, the culture ratio 106:106 showed the highest level of total concentrations of all different kinds of flavor compounds. Correlation analyses showed that 12 flavor compounds, including 4 fatty acids and their 2 corresponding esters, 1 terpene, and 5 aromatic compounds, that could only be produced by S. cerevisiae were significantly correlated with the initial inoculation amount of B. licheniformis. These metabolic changes in S. cerevisiae were not only a benefit for liquor aroma, but may also be related to its inhibition effect in mixed culture. This study could help to reveal the microbial interactions in Chinese liquor fermentation and provide guidance for optimal arrangement of mixed culture fermentation systems.
Co-reporter:Shanshan Wang, Yao Nie, Yan Xu, Rongzhen Zhang, Tzu-Ping Ko, Chun-Hsiang Huang, Hsiu-Chien Chan, Rey-Ting Guo and Rong Xiao
Chemical Communications 2014 vol. 50(Issue 58) pp:7770-7772
Publication Date(Web):01 May 2014
DOI:10.1039/C4CC01752H
Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs' stereoselectivity.
Co-reporter:Rong Zhang, Qun Wu, and Yan Xu
Journal of Agricultural and Food Chemistry 2014 Volume 62(Issue 33) pp:8302-8307
Publication Date(Web):July 26, 2014
DOI:10.1021/jf502053g
Nonvolatile compounds play important roles in the quality of alcoholic beverages. In our previous work, a type of cyclooctapeptide lichenysin was newly identified in Chinese strong-aroma type liquor. In this work, it was found that lichenysin could selectively affect aroma volatility in strong-aroma type (Jiannanchun) liquor. Interaction of lichenysin and volatile phenolic compounds (off-odors in strong-aroma type liquor) was characterized using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS). HS-SPME results indicated that lichenysin very efficiently suppressed the volatility of phenolic compounds by 36–48% (P < 0.05). Thermodynamic analysis showed that the binding process was mainly mediated by hydrogen bonding. Furthermore, the mixture of lichenysin and 4-ethylguaiacol revealed intermolecular cross peaks between the aH (Val) of lichenysin and the 1H of 4-ethylguaiacol, by using nuclear Overhauser effect spectroscopy. This study will help to further understand the interaction mechanisms between flavor and nonvolatile matrix components in Chinese liquors.
Co-reporter:Shanshan Wang;Yao Nie;Xu Yan;Tzu-Ping Ko;Chun-Hsiang Huang;Hsiu-Chien Chan;Rey-Ting Guo ;Rong Xiao
Acta Crystallographica Section F 2014 Volume 70( Issue 6) pp:800-802
Publication Date(Web):
DOI:10.1107/S2053230X1400908X
The NADH-dependent (R)-carbonyl reductase from Candida parapsilosis (RCR) catalyzes the asymmetric reduction of 2-hydroxyacetophenone (HAP) to produce (R)-1-phenyl-1,2-ethanediol [(R)-PED], which is used as a versatile building block for the synthesis of pharmaceuticals and fine chemicals. To gain insight into the catalytic mechanism, the structures of complexes of RCR with ligands, including the coenzyme, are important. Here, the recombinant RCR protein was expressed and purified in Escherichia coli and was crystallized in the presence of NAD+. The crystals, which belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.64, b = 106.11, c = 145.55 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.15 Å resolution. Initial model building indicates that RCR forms a homotetramer, consistent with previous reports of medium-chain-type alcohol dehydrogenases.
Co-reporter:Chao Kang;Xiao-Wei Yu
Journal of Industrial Microbiology & Biotechnology 2014 Volume 41( Issue 1) pp:49-55
Publication Date(Web):2014 January
DOI:10.1007/s10295-013-1378-z
A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca2+ but inhibited by EDTA (10 mM) and Cu2+. The Km and kcat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein.
Co-reporter:Wen-Bo Chen;Yao Nie;Xiao-Qing Mu;Wei Yan
Applied Biochemistry and Biotechnology 2014 Volume 174( Issue 7) pp:2516-2526
Publication Date(Web):2014 December
DOI:10.1007/s12010-014-1201-y
Degeneration of engineered strains and decreased production of target gene products were often observed during recombinant bioprocess. Although several strategies have been developed to ensure high levels of target gene products, these methods seemed to be complex and laborious. By investigating possible factors contributing to the decreased yield, degeneration of host cells was identified as the main cause. Based on the principle of auto-induction and the fact that the interaction between colored substrates and gene products could present visible changes, a convenient and accurate screening method was developed to evaluate the production performance of engineered strains. Due to the typicality of pullulanase from genus Klebsiella, which has been a model for the research of secretion mechanism in gram-negative bacteria, an engineered E. coli producing extracellular pullulanase was employed to illustrate the method. Consequently, according to the capability to form a colorless halo, colonies could be divided into two groups, one surrounded by clear zone and the other unable to present transparent haloes. Furthermore, the high capability of these colonies was confirmed by performing pullulanase production in liquid medium. Compared with other methods for evaluating engineered strains, the visible screening technique was suggested to have the advantages of effectiveness and accuracy.
Co-reporter:Naiqiang Li;Howard Chou;Lijun Yu
Biotechnology and Bioprocess Engineering 2014 Volume 19( Issue 6) pp:965-972
Publication Date(Web):2014 November
DOI:10.1007/s12257-014-0352-6
Cadaverine, as a biogenic amine, is an important platform chemical for the production of industrial polymers, such as polyamides, polyurethanes, and nylon. Previous efforts focused on the bio-based production of cadaverine using two lysine decarboxylases of Escherichia coli CadA and LdcC. In this study, we report the biotransformation of cadaverine using a lysine decarboxylase from Klebsiella oxytoca. Codon optimization of the gene encoding this enzyme was carried on for the heterologous expression in E. coli, which led to a system that converted more than 24% lysine-HCl to cadaverine compared to the same system expressing CadA. The system was further optimized by using three different inducible promoters to control the expression of lysine decarboxylase gene of K. oxytoca in E. coli. The final optimized system converted lysine-HCl to cadaverine at a conversion rate of 0.133%/min/g. When the optimized system described above is coupled to an industrial process, the combined process has the potential to produce cadaverine with high conversion efficiency (46%) from sugar.
Co-reporter:Wen-Bo Chen;Yao Nie;Rong Xiao
Bioprocess and Biosystems Engineering 2014 Volume 37( Issue 4) pp:601-608
Publication Date(Web):2014/04/01
DOI:10.1007/s00449-013-1026-z
Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl β-d-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml−1) and cell biomass (7.95 g DCW l−1). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 °C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular pullulanase. Finally, with a view to releasing more intracellular pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml−1, nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.
Co-reporter:Yuxia Wang, Chao Zhang, Jiming Li, Yan Xu
Food Chemistry 2013 Volume 140(1–2) pp:245-254
Publication Date(Web):1–15 September 2013
DOI:10.1016/j.foodchem.2013.02.044
The effects of three β-glucosidases (BG1, BG2 from Trichosporon asahii, AS from Aspergillus Niger) on the aroma profiles of Cabernet Gernischt (CG) were investigated, coupled with an exploration of the possible reasons for the different performances of β-glucosidases under the two different conditions (hydrolysis of grape glycoside extract and wine-making). The analysis of headspace solid-phase micro-extraction and gas chromatography–mass spectrometry revealed that volatile flavour compounds in the β-glucosidase-treated samples were significantly increased. Specially, the wines treated with β-glucosidase BG1 occupied the highest concentrations of 19 out of 23 volatile compounds that exhibited significant differences. The investigation of the effects of pH or glucose on β-glucosidases showed that low pH is the main factor that exerts a more critical and irreversible influence on the activities and structures of β-glucosidase proteins. The stronger resistances to pH and glucose provided β-glucosidase BG1 a better ability in hydrolysing aromatic precursors than other enzymes under winemaking conditions. With the HPLC analysis, eight anthocyanins were identified from CG wine. Among the three β-glucosidases, BG1 showed the lowest influence on the main anthocyanin glycosides. These results suggested that the β-glucosidase BG1 may have some potential values to complement wine quality during the winemaking process.Highlights► Aroma compounds were significantly increased in the β-glucosidase-treated samples. ► β-Glucosidase BG1 showed better performance on aroma improvement under winemaking conditions. ► The effects of pH and glucose on enzymes were investigated for the performance differences. ► β-Glucosidase BG1 exhibited the lowest influence on the main anthocyanin glycosides.
Co-reporter:Shuang Chen, Yan Xu, and Michael C. Qian
Journal of Agricultural and Food Chemistry 2013 Volume 61(Issue 47) pp:11295-11302
Publication Date(Web):October 5, 2013
DOI:10.1021/jf4030536
The aroma profile of Chinese rice wine was investigated in this study. The volatile compounds in a traditional Chinese rice wine were extracted using Lichrolut EN and further separated by silica gel normal phase chromatography. Seventy-three aroma-active compounds were identified by gas chromatography–olfactometry (GC-O) and gas chromatography–mass spectrometry (GC-MS). In addition to acids, esters, and alcohols, benzaldehyde, vanillin, geosmin, and γ-nonalactone were identified to be potentially important to Chinse rice wine. The concentration of these aroma-active compounds in the Chinese rice wine was further quantitated by combination of four different methods, including headsapce–gas chromatography, solid phase microextraction–gas chromatography (SPME)-GC-MS, solid-phase extraction–GC-MS, and SPME-GC–pulsed flame photometric detection (PFPD). Quantitative results showed that 34 aroma compounds were at concentrations higher than their corresponding odor thresholds. On the basis of the odor activity values (OAVs), vanillin, dimethyl trisulfide, β-phenylethyl alcohol, guaiacol, geosmin, and benzaldehyde could be responsible for the unique aroma of Chinese rice wine. An aroma reconstitution model prepared by mixing 34 aroma compounds with OAVs > 1 in an odorless Chinese rice wine matrix showed a good similarity to the aroma of the original Chinese rice wine.
Co-reporter:Shuang Chen, Dong Wang, and Yan Xu
Journal of Agricultural and Food Chemistry 2013 Volume 61(Issue 40) pp:9712-9718
Publication Date(Web):September 12, 2013
DOI:10.1021/jf402867m
The aroma characteristics of sweet-type Chinese rice wine were studied by sensory analysis, aroma extract dilution analysis (AEDA), and quantitative analysis. Sensory evaluation demonstrated that a caramel-like note was the most distinctive characteristic for sweet-type Chinese rice wine. AEDA was carried out on the extract of a typical sweet-type Chinese rice wine sample. Thirty-nine odor-active regions were detected in the sample with a flavor dilution (FD) factor ≥8, and 37 of these were further identified. Among them, sotolon and 2- and 3-methylbutanol showed the highest FD factor of 1024, followed by 2-acetyl-1-pyrroline (tentatively identified), dimethyl trisulfide, 2-phenylethanol, and vanillin with a FD factor of 512. Sotolon was identified as a key aroma compound in Chinese rice wine for the first time. AEDA results indicated that sotolon (caramel-like/seasoning-like) was the potentially key contributor to the caramel-like descriptor of sweet-type Chinese rice wine. The concentration of sotolon in Chinese rice wine was further quantitated by Lichrolut-EN solid-phase extraction coupled with microvial insert large volume injection method. The content of sotolon ranged from 35.93 to 526.17 μg/L, which was above its odor threshold (9 μg/L) for all Chinese rice wine samples. The highest concentration of sotolon was found in the sweet-type Chinese rice wine, which highlighted the important aroma role of sotolon for this particular type of Chinese rice wine.
Co-reporter:Hai Du, Hu Lu, Yan Xu, and Xiaowei Du
Journal of Agricultural and Food Chemistry 2013 Volume 61(Issue 6) pp:1343-1348
Publication Date(Web):January 20, 2013
DOI:10.1021/jf3040513
Diverse Streptomyces species act as geosmin producers in the Chinese liquor-making process. In this paper, the ecology of these Streptomyces species was analyzed using denaturing gradient gel electrophoresis (DGGE) of amplified Actinobacteria-specified rDNA. The result showed that Streptomyces were widely distributed during Daqu incubation, and multiple processing, geographic, and climate factors can affect their distribution and diversity. The genes associated with geosmin production were characterized in four geosmin-producing Streptomyces strains, all of which were isolated from geosmin-contaminated Daqu. On the basis of this information, a real-time PCR method was developed, enabling the detection of traces of Streptomyces in complex solid-state matrices. The primer was targeted at the gene coding for geosmin synthase (geoA). The real-time PCR method was found to be specific for geosmin-producing Streptomyces and did not show any cross-reactivity with geosmin-negative isolates, which are frequently present in the Chinese liquor-brewing process. Quantification of geoA in the Chinese liquor-making process could permit the monitoring of the level of geosmin producers prior to the occurrence of geosmin production. Comparison of the qPCR results based on the gene encoding geosmin synthase and Actinobacteria-specified rDNA showed that about 1–10% of the Actinobacteria carry the geosmin synthesis gene.
Co-reporter:Chao Kang;Xiao-Wei Yu
Journal of Industrial Microbiology & Biotechnology 2013 Volume 40( Issue 8) pp:855-864
Publication Date(Web):2013 August
DOI:10.1007/s10295-013-1284-4
A novel endoprotease Endo-Pro-Aspergillus niger (endoprotease EPR) was first successfully expressed at high level in the methylotrophic yeast Pichia pastoris and the purification procedure was established. The endoprotease EPR is 95 % identity with proline specific endopeptidase from A. niger CBS513.88 (EMBL; AX458699), while sharing low identity with those from other microorganisms. The purified endoprotease EPR was a monomer of 60 kDa. Furthermore, the peptide mass fingerprinting (PMF) analysis confirmed that the purified protein was an endoprotease Endo-Pro-Aspergillusniger. A three-dimensional model revealed that the active site of the enzyme was located in Ser(179)-Asp(458)-His(491), based on template 3n2zB with sequence identity of 17.6 %. The optimum pH and temperature of the endoprotease EPR were pH 4–5 and 35 °C, and the stabilities were pH 3–7 and 15–60 °C, respectively. Furthermore, the endoprotease EPR had the ability to digest peptides with the C-terminal of proline as well as alanine, and was also capable of hydrolyzing larger peptides. The properties of the endoprotease EPR made it a highly promising candidate for future application in the field of brewing and food process.
Co-reporter:Chong Sha;Xiao-Wei Yu;Fei Li
Applied Biochemistry and Biotechnology 2013 Volume 169( Issue 4) pp:1160-1172
Publication Date(Web):2013 February
DOI:10.1007/s12010-012-0050-9
In this work, the high-level expression of the lipase r27RCL was achieved by optimization of the lipase gene copy number in the host strain Pichia pastoris. The copy number of the lipase gene proRCL from Rhizopus chinensis CCTCC M201021 was quantified by real-time quantitative polymerase chain reaction and a range of Mut+P. pastoris strains carrying one, three, five, and six copies of proRCL were obtained. The maximum lipase activity was achieved at 12,500 U/mL by the five-copy recombinant strain after 96 h of methanol induction in the 7-L fermenter. However, the enzyme activity of the six-copy recombinant strain decreased remarkably. By transcription analysis of proRCL, ERO1, and PDI, it suggested that unfolded protein response seemed to be triggered in the highest copy recombinant strain after 24 h. Thus, elaborate optimization of foreign gene dosage was very important for the high-level expression of foreign proteins in P. pastoris.
Co-reporter:Wen-Bo Chen;Yao Nie
Applied Biochemistry and Biotechnology 2013 Volume 169( Issue 1) pp:41-54
Publication Date(Web):2013 January
DOI:10.1007/s12010-012-9948-5
A strain with the power to produce extracellular pullulanase was obtained from the sample taken from a flour mill. By sequencing its 16S rDNA, the isolate was identified as Klebsiella variicola SHN-1. When the gene encoding pullulanase, containing the N-terminal signal sequence, was cloned into Escherichia coli BL21 (DE3), extracellular activity was detected up to 10 U/ml, a higher level compared with the results in published literature. Subsequently, the recombinant pullulanase was purified and characterized. The main end product from pullulan hydrolyzed by recombinant pullulanase was determined as maltotriose with HPLC, and hence, the recombinant pullulanase was identified as type I pullulanase, which could be efficiently employed in starch processing to produce maltotriose with higher purity and even to evaluate the purity of pullulan. To investigate the effect of signal peptide on secretion of the recombinant enzyme, the signal sequence was removed from the constructed vector. However, secretion of pullulanase in E. coli was not influenced, which was seldom reported previously. By localizing the distribution of pullulanase on subcellular fractions, the secretion of recombinant pullulanase in E. coli BL21 (DE3) was confirmed, even from the expression system of nonsecretory type without the assistance of signal peptide.
Co-reporter:Jian-Feng Wu
Food Science and Biotechnology 2013 Volume 22( Issue 5) pp:1-6
Publication Date(Web):2013 October
DOI:10.1007/s10068-013-0209-3
Pyrazine compounds in Chinese liquors are one of the most important factors that affect the flavor. However, only limited information is available regarding pyrazine compounds in Chinese liquors. An analytical method for pyrazine compounds (9 pyrazines) was developed using head space solid phase microextraction (HS-SMPE) and gas chromatography with nitrogen-phosphorus detection (GC-NPD). Pyrazine compounds in different types of Chinese liquors collected from different plants were also evaluated and compared by the methods devised in this study. The linearity and recovery with this method were satisfactory in all test cases. Quantitative results revealed that concentrations of 7 pyrazines were higher in the liquors from King’s Luck than from other plants. Further research is needed on the mechanism responsible for high concentrations of pyrazine compounds in Chinese liquors.
Co-reporter:Xiang-Yang Ge;Xiang Chen
Journal of Industrial Microbiology & Biotechnology 2013 Volume 40( Issue 3-4) pp:345-352
Publication Date(Web):2013 April
DOI:10.1007/s10295-013-1233-2
This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.
Co-reporter:Chong Sha;Xiao-Wei Yu;Meng Zhang
Journal of Industrial Microbiology & Biotechnology 2013 Volume 40( Issue 11) pp:1241-1249
Publication Date(Web):2013 November
DOI:10.1007/s10295-013-1328-9
The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space–time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.
Co-reporter:Hai Du and Yan Xu
Journal of Agricultural and Food Chemistry 2012 Volume 60(Issue 9) pp:2288-2292
Publication Date(Web):February 12, 2012
DOI:10.1021/jf204648e
Geosmin is the major cause of the common earthy off-flavor in light-aroma type Chinese liquor and, thus, highly detrimental to the aromatic quality. To find out its origin, the evolving process of geosmin in light-aroma type liquor making was monitored, and microbial analysis of Daqu containing geosmin was carried out. The results showed that geosmin appeared in all the fermented sorghums at different fermentation periods. About 57% geosmin in the fermented sorghums was distilled into liquor. During the distillation process, the peak of geosmin concentration appeared when alcohol content was 50–60% vol. More importantly, high geosmin content was observed during the Daqu-making process. Furthermore, five Streptomyces strains were isolated from different types of Daqu used for the fermentation of light-aroma type liquor. All of them produced only geosmin as the main volatile metabolite but no 2-methylisoborneol (2-MIB). It appears that microorganisms developing in Daqu are responsible for the presence of geosmin in liquor. Because of the relatively low detection threshold estimated at 110 ng/L in 46 vol % hydroalcoholic solution, the presence of geosmin in Daqu may pose a risk for Chinese liquor producers.
Co-reporter:Qun Wu and Yan Xu
Journal of Agricultural and Food Chemistry 2012 Volume 60(Issue 8) pp:2033-2038
Publication Date(Web):January 30, 2012
DOI:10.1021/jf204270h
Although Maotai flavor liquor is exclusive due to its soy sauce flavor, knowledge of its key compound and production mechanism is still scarce until now. To gain insight into the production mechanism of soy sauce flavor, a soy sauce flavor producing strain with high efficiency and heat-resistant capability was obtained, and the metabolic mechanism of the strain was investigated with the technique of microarray profiling. Because high temperature was a key factor for soy sauce flavor production, the global gene expression of this heat-resistant strain fermented at 55 °C was analyzed. Except for the responsive increase of heat shock proteins, which maintained cell survival during heat stress, biosynthesis of cysteine was also up-regulated. In addition, some metabolites were significantly increased when cysteine was added to the fermentation medium, such as 2,3-butanediol, 3-hydroxy-2-butanone, and tetramethylpyrazine, which were important flavor compounds in soy sauce flavor liquor and might be related with soy sauce flavor. The results indicated that cysteine might play an important role in the formation of soy sauce flavor compound, and it might act as an indirect precursor or stimulator of soy sauce flavor formation. This was the first use of the microarray profiling tool to investigate the fermentative strains for Chinese traditional liquor, which would allow a deeper insight into the mechanism of the formation of soy sauce flavor compound.
Co-reporter:Jun Liu;Yao Nie;Guang-ao Zhao
Bioprocess and Biosystems Engineering 2012 Volume 35( Issue 4) pp:651-657
Publication Date(Web):2012 May
DOI:10.1007/s00449-011-0643-7
Urea in alcoholic beverages is a precursor of ethyl carbamate, which is carcinogenic. Acid urease (EC 3.5.1.5) is regarded as a good approach to scavenge the urea. The acid urease of Enterobacter sp. R-SYB082, with lower optimum pH than the widely used commercial acid urease, exhibited a urea removal rate of 66.5% in Chinese rice wine, which was higher than that of the commercial acid urease (58.9%). The production of the acid urease was optimized from 1,100 to 2,504 U L−1 by an approach which includes the optimization of initial glucose concentration, the elevation of anaerobic level of the reactor by charging CO2 and in vitro natural activation of the target enzyme by simple cold storage (4°C). These would open up the possibility for developing industrial application of this acid urease for producing high-quality Chinese rice wine.
Co-reporter:Yao Nie, Rong Xiao, Yan Xu and Gaetano T. Montelione
Organic & Biomolecular Chemistry 2011 vol. 9(Issue 11) pp:4070-4078
Publication Date(Web):20 Apr 2011
DOI:10.1039/C0OB00938E
The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potential. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes.
Co-reporter:Yuxia Wang, Jiming Li, and Yan Xu
Journal of Agricultural and Food Chemistry 2011 Volume 59(Issue 20) pp:11219-11227
Publication Date(Web):September 14, 2011
DOI:10.1021/jf203693v
Two novel β-glucosidases from Trichosporon asahii, named BG1 and BG2, were purified to electrophoretic homogeneity using ammonium sulfate precipitation, hydrophobic interaction, ion exchange, and gelfiltration chromatography. The molecular weight of BG1 and BG2 were estimated as 160 kDa and 30 kDa, respectively. The Km, Vmax, Kcat, and Kcat/Km values of the two β-glucosidases for p-nitrophenyl-β-d-glucopyranoside were determined. Both enzymes showed relatively high affinity to p-nitrophenyl-β-d-glucopyranoside in 4-nitrophenol glycosides and gentiobiose in saccharide substrates. The enzymes exhibited optimum activity at pH 6.0 and pH 5.5, respectively. Their respective optimum temperatures were 70 and 50 °C. Metal ions and inhibitors had different effects on the enzymes activities. Circular dichroism (CD) spectroscopy demonstrated that the purified BG1 exhibited a β-sheet-rich structure and that BG2 displayed a high random coil conformation. HPLC analysis of transglycosylation and reverse hydrolysis assays revealed that only BG1 possessed transglycosylation activity and synthesized cello-oligosaccharides by the addition of glucose. This suggested that BG1 could be used to produce complex bioactive glycosides and could be considered as a potential enzyme for industrial application.
Co-reporter:Hai Du, Wenlai Fan, and Yan Xu
Journal of Agricultural and Food Chemistry 2011 Volume 59(Issue 15) pp:8331-8337
Publication Date(Web):June 11, 2011
DOI:10.1021/jf201171b
Earthy odor is one of the most frequent and serious causes for the aroma deterioration in Chinese liquor, which causes a dirty and dusty impression. The odor in Chinese liquor is similar to that of rice husk, one kind of auxiliary material widely used as a filler in the distillation process. So it is experientially hypothesized that such odor may derive from rice husk. In this paper, the gas chromatography–olfactometry (GC–O) technique and gas chromatography–mass spectrometry (GC–MS) were used to discover and identify the characteristic odoriferous zone of Chinese liquor marked by earthy odor. Geosmin was found to be responsible for this odor. The levels of the compound in ten bottled liquors and thirty liquors aging for different years belonging to four different aroma types were determined by the optimized headspace–solid phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS) method. Quantitative analysis of bottled liquor revealed the presence of geosmin in all aroma type liquors with concentrations ranging from 1.10 μg/L to 9.90 μg/L, except for strong-aroma type liquor. Meanwhile in the aged liquors belonging to the same aroma type, geosmin was detected with significant concentrations and high odor activity values (OAVs) during different years of aging. However, geosmin was not detected in steamed rice husk nor in nonsteamed rice husk, which suggests that rice husk is not the origin of earthy odor in Chinese liquor, and there may be another origin of it during the brewing process.
Co-reporter:Zhen Yan, Yao Nie, Yan Xu, Xiang Liu, Rong Xiao
Tetrahedron Letters 2011 Volume 52(Issue 9) pp:999-1002
Publication Date(Web):2 March 2011
DOI:10.1016/j.tetlet.2010.12.069
A simple, highly efficient, and economical biphasic cell-free system was developed for biocatalytic reduction of prochiral aromatic ketones to furnish enantiopure alcohols. This system is characterized by using endogenous enzymes of the cell-free extract to form enzyme-coupled NADPH recycling system. Besides, it offered much higher productivity than whole cells and greatly simplified the preparation process of biocatalysts in comparison with isolated enzymes. Various prochiral aromatic ketones, especially α-substituted acetophenone derivatives, were reduced to chiral alcohols with excellent enantiomeric excess (ee) and moderate to good yield by this cell-free system.
Co-reporter:Xinliang Mo, Yan Xu and Wenlai Fan
Journal of Agricultural and Food Chemistry 2010 Volume 58(Issue 4) pp:2462-2469
Publication Date(Web):January 20, 2010
DOI:10.1021/jf903631w
The aroma of Chinese rice wine Qu is one of the most important factors that influences the flavor of Chinese rice wine. To better understand the aroma of Qu, aroma compounds in four wheat Qus and two xiao Qus were identified by chromatography−olfactometry (GC-O) after solvent extraction followed by solvent-assisted flavor evaporation (SAFE). A total of 39 aroma compounds were characterized by GC-O. On the basis of aroma intensity, 1-hexanal, ethyl hexanoate, 1-octen-3-ol, and phenylacetaldehyde were found to be the most important aroma compounds in all six Qus. In addition, 3-methylbutanol and 2-phenylethanol also played an important role in the aroma of two xiao Qus. Headspace solid-phase microextraction (HS-SPME) was used for quantifying aroma compounds identified in the Qus. The method enabled limits of detection and quantification of <40.8 and <136.0 μg/L, respectively. Linearity and recovery were satisfied in all cases. Quantitative analysis revealed that volatiles of six Qus had a wide range of concentration. Principal component analysis applied to the data differentiated the six Qus well.
Co-reporter:Qingsen Hu;Yao Nie
Journal of Chemical Technology and Biotechnology 2010 Volume 85( Issue 2) pp:252-257
Publication Date(Web):
DOI:10.1002/jctb.2296
Abstract
BACKGROUND: Microbial stereoinversion has been widely used for the biosynthesis of numerous chiral compounds. However, little work has been done to improve the efficiency of microbial stereoinversion. This study investigated the bottleneck in the deracemization of 1-phenyl-1,2-ethanediol (PED), and then the efficiency and the sustainability of biocatalyst was improved significantly by using a strategy.
RESULTS: When (S)-PED concentration exceeded 17.5 g L−1, it strongly inhibited deracemization. Furthermore, the deficiency of NADPH regeneration also limited such reaction. To overcome these limitations, extractive biocatalysis was developed using adsorbent resin NKII combined with xylose addition for cofactor regeneration. Compared with the initial reaction condition, which only afforded (S)-PED with 35% optical purity after the first batch reaction at 30 g L−1 substrate concentration, the cells in the new system could be reused three times and the optical purity remained at a high level of 95%.
CONCLUSION: Product inhibition and coenzyme regeneration had a significant effect on catalytic activity of Candida parapsilosis. By using a resin and D-xylose, the efficiency and reusability of whole-cell catalyst can be considerably improved, which would be helpful for effective synthesis of high value chiral intermediates. Copyright © 2009 Society of Chemical Industry
Co-reporter:Bing-Feng Zhu
Journal of Industrial Microbiology & Biotechnology 2010 Volume 37( Issue 8) pp:815-821
Publication Date(Web):2010 August
DOI:10.1007/s10295-010-0726-5
The effects of initial culture pH ranging from 5.0 to 7.5 on biomass content, precursor 3-hydroxy-2-butanone (HB) accumulation, and 2,3,5,6-tetramethylpyrazine (TTMP) formation by Bacillus subtilis CCTCC M 208157 were investigated in shake flask fermentation. Weak acidic conditions were found to favor cell growth and precursor HB accumulation, while TTMP could be synthesized more efficiently in conditions with initial pH towards neutrality. Batch bioprocess of TTMP fermentation by Bacillus subtilis CCTCC M 208157 at various controlled pH values ranging from 5.5 to 7.0 was then examined in 7.5-l fermentor. The results suggested that optimum pH for cell growth and precursor HB accumulation was 5.5 with maximum cell growth rate (Qx) and precursor HB accumulation rate (QHB) of 0.833 g l−1 h−1 and 1.118 g l−1 h−1, respectively, while optimum pH for TTMP formation was 7.0 with maximum TTMP formation rate (QTTMP) of 0.095 g l−1 h−1. A pH-shifted strategy was accordingly developed to improve TTMP production in bioreactor fermentation by shifting the culture pH from 5.5 to 7.0 after 48 h of cultivation. By applying the strategy, final TTMP concentration of 7.43 g l−1 was obtained, being 22.2% greater than that of constant-pH fermentation.
Co-reporter:Fantao Meng
Biotechnology Letters 2010 Volume 32( Issue 4) pp:533-537
Publication Date(Web):2010 April
DOI:10.1007/s10529-009-0194-z
An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length ≥6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45°C and at pH 8.0.
Co-reporter:Bing-Feng Zhu;Wen-Lai Fan
Journal of Industrial Microbiology & Biotechnology 2010 Volume 37( Issue 2) pp:179-186
Publication Date(Web):2010 February
DOI:10.1007/s10295-009-0661-5
A spore-forming Bacillus sp. was isolated from a high-temperature Daqu, a starter culture of Chinese Maotai-flavor liquor, using an endogenous precursor screening strategy. The Bacillus sp. was capable of producing a high level of 2,3,5,6-tetramethylpyrazine (TTMP) via a precursor of 3-hydroxy-2-butanone (HB). The strain was characterized as Bacillus subtilis based on morphological, physiological, and biochemical properties as well as on partial 16S rRNA gene sequences. Different carbon and nitrogen sources as well as fermentation conditions were investigated. Optimization tests showed that oxygen supply and fermentation temperature were the most important parameters determining the production process. The production of >4.08 g/l TTMP was achieved together with a high level of endogenous precursor HB accumulation (>20 g/l) in both flask and fermentor cultures when the optimized medium and cultivation conditions were applied. Our data demonstrates the effectiveness of the endogenous precursor strategy for screening microorganisms that produce flavor compounds with structure-related precursors. The high yield of TTMP and the inexpensiveness of the agro-industrial product used as the substrate (soybean meal) indicate the potential of this process for industrial application.
Co-reporter:Bing-Feng Zhu
Bioprocess and Biosystems Engineering 2010 Volume 33( Issue 8) pp:953-959
Publication Date(Web):2010 October
DOI:10.1007/s00449-010-0419-5
To examine the effects of ammonium salts on tetramethylpyrazine (TTMP) production by Bacillus subtilis CCTCC M 208157, different ammonium salts were tested, and diammonium phosphate (DAP) was found to have a predominant effect on stimulating TTMP synthesis. The DAP requirements for TTMP production were then investigated, experimental results showed that higher concentrations of DAP favored TTMP production, while both the ammonium and phosphate ions exhibited inhibitory effects on the cell growth and precursor 3-hydroxy-2-butanone accumulation. Based on the results above, a DAP feeding strategy was developed and verified in further experiments. By applying the proposed fed-batch strategy, the maximum TTMP concentrations reached 7.46 and 7.34 g/l in flask and fermenter experiments, increased by 55.1 and 29.0% compared to that of the batch TTMP fermentation, respectively. To our knowledge, these results, i.e., TTMP yields in flask or fermenter fermentations, were new records on TTMP fermentation by B. subtilis.
Co-reporter:Min-wei Xu;Xiao-qing Mu
Bioprocess and Biosystems Engineering 2010 Volume 33( Issue 3) pp:367-373
Publication Date(Web):2010 March
DOI:10.1007/s00449-009-0334-9
An extractive biocatalytic method of aqueous two-phase system was employed for stereoinversing (R)-1-phenyl-1,2-ethanediol into (S)-1-phenyl-1,2-ethanediol by Candida parapsilosis CCTCC M203011. It was observed that substrate and product inhibitions in microbial stereoinversion through one-pot oxidation and reduction were removed efficiently by extractive biocatalysis in aqueous two-phase system with PEG 4000/phosphate potassium system, and that the substrate concentration was enhanced from 15 to 30 g/L with product optical purity of 99.02% e.e. and yield of 90% after 60 h. Simultaneously, it was observed that change in cell morphology impedes the further enhancement of substrate concentration in this system but can be reversibly changed after stereoinversion or cultivation in systems without PEG.
Co-reporter:Jin-Ling Guo;Xiao-Qing Mu
Bioprocess and Biosystems Engineering 2010 Volume 33( Issue 7) pp:797-804
Publication Date(Web):2010 September
DOI:10.1007/s00449-009-0401-2
The enantioselective reduction of methyl benzoylformate to (R)-methyl mandelate, an important pharmaceutical intermediate and a versatile resolving agent, was investigated in this study. After minimizing the reaction-specific constraints (constraints dependent on the nature of the substrate and product) by preliminary selection of the reaction parameters, an effective whole cell biocatalyst (Saccharomyces cerevisiae AS2.1392) was obtained by simple screening procedures. Under further optimized conditions, a product concentration of 103 mmol L−1 could be attained within 5 h with a yield of 85.8% and an enantiometric excess of 95.4%, indicating S. cerevisiae AS2.1392 an efficient biocatalyst for the asymmetric synthesis of (R)-methyl mandelate. Furthermore, resin-based in situ product removal (ISPR) technique was applied to alleviate the substrate and product inhibition or toxicity to the whole cells. The integration of newly isolated biocatalyst and proper ISPR technique provides a practical route for the preparation of optically active pharmaceutical intermediates.
Co-reporter:Jinling Guo;Xiaoqing Mu;Changge Zheng
Journal of Chemical Technology and Biotechnology 2009 Volume 84( Issue 12) pp:1787-1792
Publication Date(Web):
DOI:10.1002/jctb.2245
Abstract
BACKGROUND: Biocatalysts have gained increasing attention because of their inherent advantages over chemical catalysts. However, the poor operational stability has always prevented their broad application. In this study, (R)-mandelic acid was chosen as a model compound of alpha-hydroxy acids. The objective was to obtain a new biocatalyst with desired operational stability for the preparation of (R)-mandelic acid as well as other optically pure alpha-hydroxy acids of pharmaceutical importance.
RESULTS: Using a two-step screening strategy, Saccharomyces ellipsoideus GIM2.105 was selected as an effective biocatalyst with high enantioselectivity and remarkable operational stability. After 20 cycles of reuse, whole cells of S. ellipsoideus GIM2.105 maintained its activity, and no obvious decrease in conversion or enantiomeric excess (ee) was observed. Furthermore, effects of various reaction parameters, including pH, temperature, co-substrate (type, concentration), substrate concentration and reaction time, on the bioreduction were studied. Under optimal conditions, (R)-mandelic acid and four substituted aromatic (R)-alpha-hydroxy acids were prepared in high ee (95–>99%) and good conversion (>90%).
CONCLUSION: The high enantioselectivity, remarkable operational stability and mild reaction conditions showed S. ellipsoideus GIM2.105 to be an economical biocatalyst with great industrial application potential for the production of optically active alpha-hydroxy acids. Copyright © 2009 Society of Chemical Industry
Co-reporter:Shu Yang Sun;Dong Wang
Journal of Chemical Technology and Biotechnology 2009 Volume 84( Issue 3) pp:435-441
Publication Date(Web):
DOI:10.1002/jctb.2058
Abstract
BACKGROUND: Purification and characterization of an intracellular lipase produced by Rhizopus chinenesis cultured in solid-state fermentation was investigated. The potential application in concentrating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil by the pure enzyme was also studied.
RESULTS: Through four successive purification steps, the enzyme was purified to homogeneity with an apparent molecular mass of 36 kDa. The lipase was active for pH between 7.0 and 9.0 and temperatures 20–45 °C. Lipase activity was slightly increased in the presence of Ca2+ and Mg2+, but strongly inhibited by Hg2+ and SDS. The pure enzyme was most active on medium chain p-nitrophenol esters, with the highest activity towards pNP-caprylate (C8). The enzyme is a non-specific lipase, because it cleaved not only the 1,3-positioned ester bonds but also the 2-positioned bond in triolein. High EPA (17.6%) and DHA (32.9%) contents were achieved using the pure lipase (100 U) within 10 h.
CONCLUSION: The enzymatic activity of the lipase on a wide variety of substrates and its stability in the presence of some organic solvents suggest that the lipase should be investigated for a range of commercial applications. The pure lipase was proved to possess potential ability for the production and concentration of EPA and DHA from fish oil. Copyright © 2008 Society of Chemical Industry
Co-reporter:Yao Nie;Teng Fei Lv;Rong Xiao
Journal of Chemical Technology and Biotechnology 2009 Volume 84( Issue 3) pp:468-472
Publication Date(Web):
DOI:10.1002/jctb.2070
Abstract
BACKGROUND: Microbial cells have been used widely in biosynthesis of chiral alcohols. However, research concerning the effect of oxygen supply in cultivation on biocatalytic activity of whole cells in organic synthesis is limited. This study improved the reaction efficiency of Candida parapsilosis catalyzing (R,S)-1-phenyl-1,2-ethanediol (PED) deracemization by controlling agitation during cell cultivation.
RESULTS: The increase of dissolved oxygen concentration by adjusting agitation speed from 200 to 300 rpm at aeration rate 1.5 vvm significantly improved the cell growth of C. parapsilosis and the activities of two key enzymes involved in deracemization. (S)-PED with higher optical purity of 98.23%e.e. and yield of 82.94% was formed. Compared with the initial fermentation conditions at aeration rate 0.75 vvm and agitation speed 200 rpm, enhanced oxygen supply conditions afforded better cells for highly efficient conversion under higher substrate concentration.
CONCLUSION: Oxygen supply had a significant effect on cell growth and catalytic activity of C. parapsilosis catalyzing asymmetric oxidoreduction. By conveniently controlling agitation in cultivation, cell activity and key enzymes production for a complex reaction of concurrent tandem oxidation and reduction processes can easily be conducted, which could help to cultivate cells catalyzing synthesis of interested chiral compounds. Copyright © 2008 Society of Chemical Industry
Co-reporter:Yun Teng;Dong Wang
Bioprocess and Biosystems Engineering 2009 Volume 32( Issue 3) pp:397-405
Publication Date(Web):2009 April
DOI:10.1007/s00449-008-0259-8
In order to control suitable mycelium morphology to obtain high lipase productivity by Rhizopus chinensis in submerged fermentation, the effects of fungal morphology on the lipase production by this strain both in shake flask and fermentor were investigated. Different inoculum level and shear stress were used to develop distinctive morphologies. Analyses and investigations both on micromorphology and macromorphology were performed. Study of micromorphology reveals that micromorphologies for dispersed mycelia and aggregated mycelia are different in cell shape, biosynthetic activity. Macromorphology and broth rheology study in fermentor indicate that pellet formation results in low broth viscosity. Under this condition, the oil can disperse sufficiently in broth which is very important for lipase production. These results indicate that morphology changes affected the lipase production significantly for R. chinensis and the aggregated mycelia were suggested to achieve high lipase production.
Co-reporter:HAI YAN WANG;LI FAN ;YAO GUANG JIN
Journal of Food Biochemistry 2008 Volume 32( Issue 4) pp:506-520
Publication Date(Web):
DOI:10.1111/j.1745-4514.2008.00180.x
ABSTRACT
Alcohol acetyltransferase (AAT) catalyzing 2-phenylethyl alcohol and acetyl co-enzyme A (acetyl-CoA) to form phenylethyl acetate in Hanseniaspora valbyensis was studied. The enzyme was solubilized from lipid particles using 1% Triton X-100 and then purified by three steps of chromatographic separations with diethylaminoethlyl (DEAE) Sepharose, Sephadex G-75 and Octyl Sepharose, respectively. Its molecular weight was estimated to be about 37 kDa. It was discovered that AAT was most active at pH 7.0 and 30C and was stable between pH 7.0–8.0. But most activity was lost at temperatures above 10C. It was found that AAT was strongly inhibited by heavy metal ions such as Hg2+, Zn2+and Pb2+, slightly stimulated by Mg2+and slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and Mn2+. The Km was 12.7 mM for 2-phenylethyl alcohol and 23.2 mM for isoamyl alcohol. Finally, the enzyme was observed to catalyze the esterification of other alcohols with acetyl-CoA, but have preference for 2-phenylethyl alcohol.
PRACTICAL APPLICATIONS
Aroma and flavor are affected by the esters that exist in cider. Alcohol acetyltransferase (AAT) can catalyze the formation of esters. Moreover, Saccharomyces cerevisiae and non-Saccharomyces yeast produce diverse esters with different concentration during fermentation. The influence of non-Saccharomyces yeast on the sensory quality of wine and mixed fermentation using different yeasts have been discussed. So, the purification of AAT from Hanseniaspora valbyensis provides an understanding of the formation of esters in non-Saccharomyces yeast.
Co-reporter:Yuping ZHAO, Jiming LI, Yan XU, Hui DUAN, Wenlai FAN, Guang'ao ZHAO
Chinese Journal of Chromatography 2008 Volume 26(Issue 2) pp:212-222
Publication Date(Web):March 2008
DOI:10.1016/S1872-2059(08)60014-0
A method for the preparation of volatile compounds in Changyu XO brandy was established. The volatile compounds were extracted using liquid-liquid extraction and then were separated into two fractions, namely, the acidic/water-soluble fraction and the neutral/basic fraction. The neutral/basic fraction was furthermore separated into 4 fractions using silica gel normal phase chromatography, and each fraction was then concentrated and analyzed using gas chromatography-mass spectrometry (GC-MS). In comparison with the pure standards and the retention indices (RIs) reported in the literature, a total of 302 volatile compounds were identified in Changyu XO brandy, including 30 alcohols, 35 aldehydes and ketones, 20 carboxylic acids, 104 esters, 24 substituted benzenes and derivatives, 14 phenolic derivatives, 14 acetals, 16 furan derivatives, 22 terpenic and norisoprenoid derivatives, and 23 others. It was demonstrated that this method of preparation was effective for the separation and concentration of volatile compounds in Changyu XO brandy.
Co-reporter:Hai-Yan Wang;Xiao-Jun Zhang;Li-Ping Zhao
Journal of Industrial Microbiology & Biotechnology 2008 Volume 35( Issue 6) pp:603-609
Publication Date(Web):2008 June
DOI:10.1007/s10295-008-0323-z
Bacterial populations in fermented grains during fermentation may play important roles in Chinese liquor flavor. PCR-based denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene library analysis were performed to analyze the bacterial community structure of two styles of liquor. The results of DGGE profiles showed that bacterial diversity decreased with the fermentation process and Lactobacillus acetotolerans became the predominant species at the end of the fermentation. But the obvious differences of bacterial community appeared in the middle stage of two styles of liquor fermentation, in which the different upstream production techniques were used. Moreover, 16S rRNA gene libraries of two styles were constructed. A total of 125 and 107 clones, chosen from two libraries, were grouped into 46 and 49 operational taxonomic units (OTUs) by amplified ribosomal DNA restriction analysis. According to sequencing results of clones, the predominant bacteria in strong aroma style fermented grains were those from the class Bacilli, Bacteroidetes, and Clostridia, whereas the predominant bacteria in fermented grains of roasted sesame aroma style belonged to Bacilli, Flavobacteria, and Gammaproteobacteria. Molecular analysis of the bacterial diversity of the liquor fermentation will benefit the analysis of important microorganisms playing key roles in the formation of liquor flavor components.
Co-reporter:Wenlai Fan, Yan Xu and Yanhong Zhang
Journal of Agricultural and Food Chemistry 2007 Volume 55(Issue 24) pp:9956-9962
Publication Date(Web):October 31, 2007
DOI:10.1021/jf071357q
Pyrazines are very important impact aroma compounds in Chinese liquors. The identification of pyrazine derivatives was carried out by liquid–liquid extraction (LLE). The liquor sample was adjusted to the H+ concentration of 1 N with 12 N HCl and then concentrated by rotatory evaporator under vacuum condition. The concentrated liquor was extracted by diethyl ether, and the residual aqueous phase was adjusted to pH 10. The basic compounds were detected and identified by gas chromatography (GC)–mass spectrometry (MS). A total of 27 pyrazines were identified in Chinese liquors, mainly alkyl- and acetylpyrazines. A method for determining pyrazines in Chinese liquors was developed. It involves extraction by headspace (HS) solid phase microextraction (SPME) and determination using GC–flame thermionic detector (FTD). The optimum method was that the sample alcohol concentration was diluted to 12% vol by freshly redistilled–deionized water, and the diluted samples were saturated with NaCl and equilibrated at 50 °C for 15 min and extracted for 30 min at the same temperature. The developed method enabled detection limits of <200 ng/L. Linearity (R2 > 0.99) and recovery rate were satisfied in all cases. Pyrazines of 12 commercial typical Chinese liquors were quantified by HS-SPME followed by GC-FTD and had a wide range of concentration.
Co-reporter:Yaohui Li, Rongzhen Zhang, Yan Xu, Rong Xiao, Lei Wang, Xiaotian Zhou, Hongbo Liang, Jiawei Jiang
Process Biochemistry (September 2016) Volume 51(Issue 9) pp:1175-1182
Publication Date(Web):1 September 2016
DOI:10.1016/j.procbio.2016.05.026
•Homologous expression of SCRII was successful in C. parapsilosis/pCP-scrII.•Biotransformation efficiency of (S)-PED was significantly improved by in situ expressed SCRII.•C. parapsilosis/pCP-scrII tolerated a wide range of temperatures and substrate concentrations.•Large-scale of ketone reductions by C. parapsilosis/pCP-scrII was demonstrated.(S)-carbonyl reductase II (SCRII), a short-chain alcohol dehydrogenase from Candida parapsilosis M203011, catalyzes the bioreduction of 2-hydroxyacetophenone (2-HAP) to (S)-1-phenyl-1,2-ethanediol ((S)-PED). When SCRII was expressed in Escherichia coli, the biotransformation efficiency of (S)-PED was low. To improve its biocatalytic efficiency, the homologous expression of SCRII in C. parapsilosis M203011 was attempted. The scrII gene was cloned into an expression vector pCP carrying MAL2 as its promoter and SAT1 as its selection marker. Data obtained in this study showed that SCRII was successfully expressed in recombinant strain C. parapsilosis/pCP-scrII. The reductive activity toward 2-HAP exhibited about 2-fold and 6-fold increase in the cell-free extracts of C. parapsilosis/pCP-scrII than those of the wild-type and E. coli/pET28-SCRII. Under the optimal bioreaction conditions (pH 5.5, 35 °C), the optical purity and yield of (S)-PED were both over 99.9% produced by C. parapsilosis/pCP-scrII. Additionally, 500-mL preparative scale bioreduction with efficient whole-cell process was performed, and the optical purity was over 99.9% with an isolated yield of about 70%. Our work not only demonstrated the high catalytic efficiency given by the homologous expression of SCRII in C. parapsilosis, but also provides an economical method for the preparation of optically pure chiral alcohols with whole-cell process.The homologously expressed SCRII in C. parapsilosis gave good performance on asymmetric reduction of 2-HAP to (S)-PED.Download high-res image (155KB)Download full-size image
Co-reporter:Guangyuan Jin, Yang Zhu, Yan Xu
Trends in Food Science & Technology (May 2017) Volume 63() pp:18-28
Publication Date(Web):1 May 2017
DOI:10.1016/j.tifs.2017.02.016
•Traditional Chinese liquor fermentation faces industrial challenge of modernisation.•Traditional liquor fermentation is a treasure to explore for quality of life today.•Hidden knowledge is valuable to study and apply in food and biotechnology sectors.•Multidisciplinary approach will accomplish innovation and improvement.BackgroundChinese liquor, a very popular fermented alcoholic beverage with thousands of years’ history in China, though its flavour formation and microbial process have only been partly explored, is facing the industrial challenge of modernisation and standardisation for food quality and safety as well as sustainability. Meanwhile, the hidden knowledge behind the complicated and somehow empirical solid-state fermentation process of Chinese liquor can enrich the food sector to improve our quality of life, and benefit other industrial sectors in the modern biomass-based technology, economy and society.Scope and approachThis review reveals the traditional fermentation process and characteristics of Chinese liquor, summarises the current study progress of flavour chemistry and responsible microbial process, and addresses future improvement and research needs. We provide here a detailed, systematic and critical review on Chinese liquor to improve the current industrial practice and serve the modern society with yet incompletely explored but useful principles.Key findings and conclusionsThe hidden knowledge behind the traditional Chinese liquor production is rich in useful principles including flavour chemistry, microbial growth, solid-state fermentation, enzyme production, biocatalysis, microbial community metabolism and process engineering. Studies in a more in-depth, systematic and practical way on this look-like empirical process to explore the scientific principles behind will definitely benefit the liquor industry in particular, and the (food) biotechnology sector in general.
Co-reporter:Xiao-Wei Yu, Le-Le Wang, Yan Xu
Journal of Molecular Catalysis B: Enzymatic (May 2009) Volume 57(Issues 1–4) pp:304-311
Publication Date(Web):1 May 2009
DOI:10.1016/j.molcatb.2008.10.002
Lipases are the most attractive enzymes for use in organic chemical processes. In our previous studies, a lipase from Rhizopus chinensis CCTCC M20102 was found to have very high ability of esterification of short-chain fatty acids with ethanol. In this study, we reported the cloning and expression of the lipase gene from R. chinensis in Pichia pastoris and characterization of the recombinant lipase. The lipase gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOX1 promoter. In the induction phase, two bands of 37 kDa and 30 kDa proteins could be observed. The amino-terminal analysis showed that the 37-kDa protein was the mature lipase (30 kDa) attached with 27 amino acid of the carboxy-terminal part of the prosequence (r27RCL). The pH and temperature optimum of r27RCL and mRCL were pH 8.5 and 40 °C, and pH 8 and 35 °C, respectively. The stability, reaction kinetics and effects of metal ions and other reagents were also determined. The chain length specificity of r27RCL and mRCL showed highest activity toward p-nitrophenyl hexanoate or glyceryl tricaproate (C6) and p-nitrophenyl acetate or glyceryl triacetate (C2), respectively. This property is quite rare among lipases and gives this new lipase great potential for use in the field of biocatalysis.
Co-reporter:Lijuan Yu, Yan Xu, Xiaowei Yu
Journal of Molecular Catalysis B: Enzymatic (May 2009) Volume 57(Issues 1–4) pp:27-33
Publication Date(Web):1 May 2009
DOI:10.1016/j.molcatb.2008.06.011
A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca2+, K+ and Mg2+, but completely inhibited by Hg2+, Cu2+, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.
Co-reporter:Yun Teng, Yan Xu, Dong Wang
Journal of Molecular Catalysis B: Enzymatic (May 2009) Volume 57(Issues 1–4) pp:292-298
Publication Date(Web):1 May 2009
DOI:10.1016/j.molcatb.2008.10.003
The fungal Rhizopus chinensis could produce several types of lipase, which were mainly intracellular. During the whole-cell lipase production by this strain in submerged fermentation, it was observed that two catalytic characteristics (hydrolytic and synthetic activity) of lipases were different with addition of lipids. The hydrolytic activity of the lipase was not induced by lipids efficaciously and could be detected regardless of whether substrate-related compounds were present. However, it was found that the induction of lipids for the synthetic activity lipase was significant, and that nearly no synthetic activity was detected while the medium contained no lipids. When only a little lipid (1 g/L) was added to medium, the synthetic activity increased sharply in the initial process of fermentation. Analysis of crude membrane-bound lipase by SDS-PAGE confirmed this induction. De novo biosynthesis of lipases, especially the lipase with synthetic activity occurred only when lipids existed. Cell growth and maltose repress the lipase production with synthetic activity, but have little influence on the lipase production with hydrolytic activity. Since the production process of mycelium-bound lipase with hydrolytic activity was different, it was reasonable to consider hydrolytic activity and synthetic activity for different application purposes. Whole-cell lipase obtained from fermentation process with high synthetic activity showed excellent catalytic ability in solvent free system on synthesis of ethylcaprylate and ethyloleate, the conversion could reach more than 90% in 5 h.
Co-reporter:Ting Jiang, Chao Kang, Xiao-Wei Yu, Yan Xu
Journal of Molecular Catalysis B: Enzymatic (March 2016) Volume 125() pp:81-87
Publication Date(Web):1 March 2016
DOI:10.1016/j.molcatb.2016.01.005
•Fusion expression is an effective strategy to improve protein secretion level.•PLA2 was successfully used as a fusion chaperon for the first time.•P. pastoris GS115 can be a suitable host for heterogenous expression.In our previous studies, a prolyl endopeptidase (PEP) gene from Aspergillus oryzae (MOH) was cloned and expressed in Pichia pastoris; however, the recombinant protein expression level of MOH was very low. In the present study, the PEP expression level was successfully improved by constructing fusion expression proteins with four fusion partners, namely, Streptomyces violaceoruber Phospholipase A2 (PLA2), cellulose-binding domain (CBD), small ubiquitin-related modifier (SUMO) and maltose binding protein (MBP). The enzyme activities of the recombinant fusion proteins CLMH, SLMH, MLMH and PLMH were increased to 3.8-, 2.7-, 4.9- and 7.4-fold compared with that of the parent MOH. Moreover, the extracellular protein content of CLMH, SLMH, MLMH, PLMH were 1.42-, 1.25-, 1.67- and 1.83-fold higher compared with that of MOH. Both PLMH and MOH showed the highest activity at pH 5.5, the highest stability at pH 6.0 and maximal activity at 40 °C.Download high-res image (68KB)Download full-size image
Co-reporter:Chao Kang, Xiao-Wei Yu, Yan Xu
Journal of Molecular Catalysis B: Enzymatic (June 2014) Volume 104() pp:64-69
Publication Date(Web):1 June 2014
DOI:10.1016/j.molcatb.2014.03.012
•We construct the Pichia pastoris expression system according to a codon-optimized endoprotease Endo-Pro-Aspergillus niger (endoprotease EPR) with high activity.•We develope the fed-batch strategy for high cell-density fermentation of the endoprotease EPR.•We study the enzyme kinetic parameters such as Km, Kcat and Kcat/Km values using different strength of the chromogenic peptide.•We find the recombinant endoprotease EPR had a significantly effective on removing the beer haze protein in our study.The prolyl endopeptidase (endoprotease Endo-Pro-Aspergillus niger, endoprotease EPR) gene from A. niger was optimized according to the codon usage bias in Pichia pastoris. It was successfully expressed using expression vector pPIC9K with high activity (620 ± 25 U/l). The recombinant optimized endoprotease EPR could work in an extremely broad temperature range and over 40% relative activity were remained in the temperature range of 15–70 °C. The optimal pH value and temperature for activity were 4.0–5.0 and 35–40 °C, respectively. The enzyme kinetic parameters Km, Kcat and Kcat/Km values towards various substrates were also determined. A fed-batch strategy was first developed for high cell-density fermentation and the enzyme activity reached 1890 U/l after cultivation in 7 l fermenter. The broad temperature range and efficient expression made this enzyme possible to apply in the food industry directly. This study also sought to investigate the effect of endoprotease EPR on removing the beer haze protein.Download full-size image
Co-reporter:Yan Zhi, Qun Wu, Yan Xu
Bioresource Technology (July 2017) Volume 235() pp:
Publication Date(Web):1 July 2017
DOI:10.1016/j.biortech.2017.03.090
•Waste DGS are suitable for value-added surfactin production.•Strain MT45 produced 1.04 g/l surfactin using DGS as carbon source.•Strain X82 showed opportune hydrolases activities and no surfactin inhibition.•MT45 and X82 were co-cultured to produce surfactin using DGS as carbon source.•3.4 g/l surfactin was obtained in the optimized co-culture fermentation.Distillers’ grains (DGS), the main waste by-products of Chinese liquor industry, were used as substrate for surfactin production. Bacillus amyloliquefaciens MT45 could grow with DGS as sole carbon source to produce 1.04 g/l surfactin. However, low amylase activity of MT45 limited sugar supply and the subsequent surfactin production. Therefore, MT45 was co-cultured with Bacillus strains that exhibited remarkable hydrolases activities. Surfactin yield increased by 50% when MT45 was co-cultured with B. amyloliquefaciens X82 that showed no product inhibition effect and did not develop extracellular matrix. The inoculation ratio of X82 greatly influenced the sugar supply, cellular growth, and surfactin production of the co-culture fermentation. Maximum surfactin titration (3.4 g/l) was obtained when MT45 and X82 were co-cultured with inoculation ratio at 1:0.5, using 200 g/l DGS. This work highlights the feasibility of using industrial waste DGS as promising feedstocks to produce value-added surfactin by co-culture fermentation.Download high-res image (127KB)Download full-size image
Co-reporter:Bin Li, Yao Nie, Xiao Qing Mu, Yan Xu
Journal of Molecular Catalysis B: Enzymatic (July 2016) Volume 129() pp:21-28
Publication Date(Web):1 July 2016
DOI:10.1016/j.molcatb.2016.04.003
•Key enzymes for redox-based deracemization were selected by systematic evaluation.•Multi-enzyme system was artificially constructed for one-pot deracemization.•Enzyme-coupled deracemization system involved cofactor self-recycling.•Optically active product was achieved by biocatalytic system and process regulation.Deracemization via oxidoreductive stereoinversion is one of the most attractive methods for the preparation of enantiomerically pure compounds. However, available enzymatic system is yet limited for efficiently catalyzing deracemization to produce optically pure alcohol in certain configuration. Through evaluation of available stereoselective oxidoreductases on activity, selectivity, and cofactor dependency, the suitable candidates were obtained to construct the enzymatic deracemization system involving cofactor self-recycling. For deracemizing (R,S)-1-phenyl-1,2-ethanediol (PED) to (R)-PED, a facile one-pot system was established by combination of two stereoselective oxidoreductases, the stereospecific carbonyl reductase 1 (SCR1) and the ketoreductase (KRD). To rebalance the activities and catalytic functions of different enzymes involved in the multi-enzyme system, the reaction conditions of SCR1-catalyzed oxidation and KRD-mediated reduction were optimized, respectively. Consequently, the deracemization system involving cofactor self-recycling was built to produce (R)-PED with the optical purity of 95.50%e.e. and the yield of 91.62% from the corresponding racemate (1 g L−1), under the optimal reaction conditions including activity ratio of SCR1/KRD 1:4 (SCR1 10 U mL−1 and KRD 40 U mL−1), molar ratio of NADP+/NADPH 3:1, 30 °C, and pH 7.0. Therefore, the developed strategy would be useful to construct the multi-enzyme deracemization system based on systematic evaluation of enzyme features.Download full-size image
Co-reporter:Rongyun Guo, Yao Nie, Xiao Qing Mu, Yan Xu, Rong Xiao
Journal of Molecular Catalysis B: Enzymatic (July 2014) Volume 105() pp:66-73
Publication Date(Web):1 July 2014
DOI:10.1016/j.molcatb.2014.04.003
•Aldo-keto reductase (AKR) toolbox was identified from Candida parapsilosis genome.•The new AKRs were active to various carbonyl compounds with high enantioselectivity.•An enzyme was gained with excellent enantioselectivity and broad substrate spectrum.•Genomic mining is powerful to develop novel AKRs for fine biochemical synthesis.Biocatalytic reduction of prochiral ketones offers significant potential in synthesis of optically active alcohols. However, so far the application of aldo-keto reductases (AKRs) in asymmetric reduction has been hampered due to limited availability of AKRs with high enantioselectivity and catalytic efficiency. Based on the genome sequence of Candida parapsilosis, a versatile bioresource for asymmetric reduction, eight open reading frames encoding putative AKRs were discovered and expressed, and the resulted enzymes (CPARs), comprising an AKR toolbox, were evaluated toward various carbonyl substrates. The CPARs were active to the selected substrates, especially 2-hydroxyacetophenone and ethyl 4-chloro-3-oxobutyrate. Additionally, most of them were obviously enantioselective to the substrates and gave alcohol products with optical purity up to 99%e.e. Of the enzymes, CPAR4 was outstanding with excellent enantioselectivity and broad substrate spectrum. All these positive features demonstrate that genomic mining is powerful in searching for novel and efficient biocatalysts of desired reactions for pharmaceuticals and fine chemicals synthesis.Download full-size image
Co-reporter:Wenyou Zhu, Yao Nie, Yan Xu
Food Control (August 2017) Volume 78() pp:222-229
Publication Date(Web):1 August 2017
DOI:10.1016/j.foodcont.2017.02.062
•A new USLE-SPE-UPLC-MS/MS method was developed to determine ochratoxin A in Daqu.•The distribution rule of OTA in Daqu was revealed for the first time.•The fermentation temperature is the key factor influencing OTA occurrence in Daqu.•Some fungi may be able to synthesize OTA when the temperature exceeds 45 °C.Daqu, a traditional starter culture mainly used to produce Chinese liquor and vinegar, is spontaneously fermented by diverse bacteria, yeasts and filamentous fungi under thermophilic condition. Therefore, mycotoxins may exist in Daqu, resulting in the contamination of end-foods. Ochratoxin A (OTA), a mycotoxin produced by certain species of Aspergillus and Penicillium, is not known whether existing in Daqu. However, specific method to detect OTA as well as OTA occurrence in Daqu has not been reported so far. With this in mind, a new method was developed to detect OTA in Daqu by the combination of ultrasound-assisted solid-liquid extraction (USLE), solid phase extraction (SPE) cleanup and UPLC-MS/MS. The USLE conditions of OTA from Daqu were optimized using Plackett-Burman (PB) design coupled with Box-Behnken (BB) design. Under the optimized conditions, no matrix effects were found, and the external standard method can be used to determine OTA in Daqu. The recoveries for spiked samples were 87–106% with the relative standard deviations (RSD) < 15%. The limits of detection and quantification were respectively 0.33 and 0.41 ppb. This approach was then applied to analyze 133 Daqu samples from different geographical regions in China, including 26 low temperature-, 33 medium temperature- and 74 high temperature-type Daqu. The results showed that OTA was detected in 66 samples with a maximum concentration of 28.87 ppb in low temperature Daqu, and the OTA incidence was on increase in the order of high temperature-, medium temperature- and low temperature-type Daqu. This implied that fermentation temperature is the key factor influencing OTA occurrence in Daqu. Moreover, there may be some fungi possessing the biosynthesis ability of OTA under high temperature environment (more than 45 °C).
Co-reporter:Wan Song, Yao Nie, Xiao Qing Mu, Yan Xu
Protein Expression and Purification (August 2016) Volume 124() pp:23-31
Publication Date(Web):1 August 2016
DOI:10.1016/j.pep.2016.04.008
•Bacillus naganoensis pullulanase (PUL) was successfully expressed in B. subtilis.•PUL expression from B. subtilis was enhanced by optimization of promoter and host.•Loss of recombinant plasmid would be main issue concerning expression stability.•Promoter and host as regulatory elements remarkably impacted on protein expression.Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml−1 from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml−1 from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml−1. Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis.
Co-reporter:Xiaoqing Wang, Xiaowei Yu, Yan Xu
Enzyme and Microbial Technology (7 August 2009) Volume 45(Issue 2) pp:94-102
Publication Date(Web):7 August 2009
DOI:10.1016/j.enzmictec.2009.05.004
The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg2+, Ca2+, Cu2+, Zn2+, Co2+, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H2O2. The enzyme had good stability in the low- and non-polar solvents.
Co-reporter:Xueshan Wang, Hai Du, Yan Xu
International Journal of Food Microbiology (6 March 2017) Volume 244() pp:27-35
Publication Date(Web):6 March 2017
DOI:10.1016/j.ijfoodmicro.2016.12.018
•This study tracked the sources of prokaryotes in fermented grain.•Daqu was the main source of aerobes and facultative aerobes.•Pit mud acted as the sustained-release source of anaerobes in fermented grain.The fermentation process of Chinese strong-flavor liquor involves numerous microbes originating from Daqu and pit mud. Daqu is the starter of fermentation, and pit mud acts as another source of inoculum of microbes in the liquor-making process. However, the contribution of microbes in pit mud and Daqu to fermented grain, and the sources of microbes in fermented grain are still waiting to be defined clearly. In this study, prokaryotic communities in fermented grain, pit mud and Daqu were identified via next generation sequencing of the V4 region of 16S rRNA gene. Principal-coordinate analysis indicated that Daqu had stronger influence on the prokaryotic communities in fermented grain at the prophase of fermentation, but pit mud influenced the fermented grain continuously during the whole fermentation process. Totally, 299 genera were detected in all fermented grain, pit mud and Daqu samples. Among them, 204 genera were detected in 3 days' fermented grain. Ten genera (Lactobacillus, Leuconostoc, Staphylococcus, Gluconobacter, Acetobacter, Petrimonas, Clostridium, Ruminococcus, Methanobacterium and Methanobrevibacter) were dominant, and accounted for 84.31%–87.13% relative abundance of the total prokaryotic community in fermented grain. Venn analysis indicated Daqu was the main source of strict aerobes and facultative aerobes, which took up over 74% of prokaryotic communities in fermented grain. Conversely, pit mud was the sustained-release source of anaerobes, which accounted for over 14% of prokaryotic communities in fermented grain. In addition, part of anaerobes originated from both Daqu and pit mud. This study could help track the source of prokaryotic communities in fermented grain, and improve the quality and controllability in liquor production.
Co-reporter:Peng Wang, Qun Wu, Xuejian Jiang, Zhiqiang Wang, Jingli Tang, Yan Xu
International Journal of Food Microbiology (5 June 2017) Volume 250() pp:59-67
Publication Date(Web):5 June 2017
DOI:10.1016/j.ijfoodmicro.2017.03.010
•B. licheniformis inoculation affected the microbial community structure.•B. licheniformis inoculation influenced the enzyme activity of Daqu.•B. licheniformis inoculation enriched pyrazines and aromatic compounds.Chinese liquor is produced from spontaneous fermentation starter (Daqu) that provides the microbes, enzymes and flavors for liquor fermentation. To improve the flavor character of Daqu, we inoculated Bacillus licheniformis and studied the effect of this strain on the community structure and metabolic profile in Daqu fermentation. The microbial relative abundance changed after the inoculation, including the increase in Bacillus, Clavispora and Aspergillus, and the decrease in Pichia, Saccharomycopsis and some other genera. This variation was also confirmed by pure culture and coculture experiments. Seventy-three metabolites were identified during Daqu fermentation process. After inoculation, the average content of aromatic compounds were significantly enriched from 0.37 mg/kg to 0.90 mg/kg, and the average content of pyrazines significantly increased from 0.35 mg/kg to 5.71 mg/kg. The increase in pyrazines was positively associated with the metabolism of the inoculated Bacillus and the native genus Clavispora, because they produced much more pyrazines in their cocultures. Whereas the increase in aromatic compounds might be related to the change of in situ metabolic activity of several native genera, in particular, Aspergillus produced more aromatic compounds in cocultures with B. licheniformis. It indicated that the inoculation of B. licheniformis altered the flavor character of Daqu by both its own metabolic activity and the variation of in situ metabolic activity. Moreover, B. licheniformis inoculation influenced the enzyme activity of Daqu, including the significant increase in amylase activity (from 1.3 g starch/g/h to 1.7 g starch/g/h), and the significant decrease in glucoamylase activity (from 627.6 mg glucose/g/h to 445.6 mg glucose/g/h) and esterase activity (from 28.1 mg ethyl caproate/g/100 h to 17.2 mg ethyl caproate/g/100 h). These effects of inoculation were important factors for regulating the metabolism of microbial communities, hence for improving the flavor profile Daqu.
Co-reporter:Qun Wu, Liangqiang Chen, Yan Xu
International Journal of Food Microbiology (2 September 2013) Volume 166(Issue 2) pp:323-330
Publication Date(Web):2 September 2013
DOI:10.1016/j.ijfoodmicro.2013.07.003
•Chinese Maotai-flavor liquor was produced by a complex yeast community.•Z. bailii, S. cerevisiae, P. membranifaciens, and S. pombe were the dominant species.•Z. bailii was a useful yeast species in Chinese Maotai-flavor liquor.•The four yeast species inhabited in different locations during fermentation.•Yeast population was consistent with sugar consumption and ethanol production.Yeasts are the most important group of microorganisms contributing to liquor quality in the solid-state fermentation process of Chinese Maotai-flavor liquor. There occurred a complex yeast community structure during this process, including stages of Daqu (the starter) making, stacking fermentation on the ground and liquor fermentation in the pits. In the Daqu making stage, few yeast strains accumulated. However, the stacking fermentation stage accumulated nine yeast species with different physio-biochemical characteristics. But only four species kept dominant until liquor fermentation, which were Zygosaccharomyces bailii, Saccharomyces cerevisiae, Pichia membranifaciens, and Schizosaccharomyces pombe, implying their important functions in liquor making. The four species tended to inhabit in different locations of the stack and pits during stacking and liquor fermentation, due to the condition heterogeneity of the solid-state fermentation, including the different fermentation temperature profiles and oxygen density in different locations. Moreover, yeast population was much larger in the upper layer than that in the middle and bottom layers in liquor fermentation, which was in accordance with the profile of reducing sugar consumption and ethanol production. This was a systematical investigation of yeast community structure dynamics in the Maotai-flavor liquor fermentation process. It would be of help to understand the fermentative mechanism in solid-state fermentation for Maotai-flavor liquor.
Co-reporter:Bi Chen, Qun Wu, Yan Xu
International Journal of Food Microbiology (2 June 2014) Volume 179() pp:80-84
Publication Date(Web):2 June 2014
DOI:10.1016/j.ijfoodmicro.2014.03.011
•A complex filamentous fungal community was observed during the liquor making process.•P. variotii and A. oryzae were the predominant species.•Stacking fermentation was essential for fungi growth and amylase accumulation.•The two species possessed high amylase activity and affected the SSF process most.Maotai-flavor liquor is produced by simultaneous saccharification and fermentation (SSF) process under solid state conditions, including Daqu (starter) making, stacking fermentation and alcohol fermentation stages. Filamentous fungi produce many enzymes to degrade the starch material into fermentable sugar during liquor fermentation. This study investigated the filamentous fungal community associated with liquor making process. Eight and seven different fungal species were identified by using culture-dependent and -independent method (PCR-denaturing gradient gel electrophoresis, DGGE) analyses, respectively. The traditional enumeration method showed that Daqu provided 7 fungal species for stacking fermentation. The total population of filamentous fungi increased from 3.4 × 103 cfu/g to 1.28 × 104 cfu/g in the first 3 days of stacking fermentation, and then decreased till the end. In alcohol fermentation in pits, the population continuously decreased and few fungal species survived (lower than 1 × 103 cfu/g) after 10 days. Therefore, stacking fermentation is an essential stage for the growth of filamentous fungi. Paecilomyces variotii, Aspergillus oryzae and Aspergillus terreus were detected by both methods, and P. variotii and A. oryzae were the predominant species. Meanwhile, P. variotii possessed the highest glucoamylase (3252 ± 526 U/g) and A. oryzae exhibited the highest α-amylase (1491 ± 324 U/g) activity among the cultivable fungal species. Furthermore, the variation of starch and reducing sugar content was consistent with the growth of P. variotii and A. oryzae in Zaopei (fermented grains) during stacking fermentation, which implied that the two filamentous fungi played an important role in producing amylase for hydrolyzing the starch.
Co-reporter:Chong Sha, Xiao-Wei Yu, Nai-Xin Lin, Meng Zhang, Yan Xu
Enzyme and Microbial Technology (10 December 2013) Volume 53(Issues 6–7) pp:438-443
Publication Date(Web):10 December 2013
DOI:10.1016/j.enzmictec.2013.09.009
•A series of strains with different gene dosage were generated in Muts phenotype.•Co-expression with PDI significantly improved the protein quantity and quality.•The highest enzyme activity was achieved by the five copy strain.•This research aids understanding the secretory pathway in Pichia pastoris.Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355 U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris.
Co-reporter:Dong Wang, Yan Xu, Tianyu Shan
Biochemical Engineering Journal (1 August 2008) Volume 41(Issue 1) pp:30-37
Publication Date(Web):1 August 2008
DOI:10.1016/j.bej.2008.03.003
Co-reporter:Yao Nie, Rong Xiao, Yan Xu and Gaetano T. Montelione
Organic & Biomolecular Chemistry 2011 - vol. 9(Issue 11) pp:NaN4078-4078
Publication Date(Web):2011/04/20
DOI:10.1039/C0OB00938E
The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potential. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes.
Co-reporter:Shanshan Wang, Yao Nie, Yan Xu, Rongzhen Zhang, Tzu-Ping Ko, Chun-Hsiang Huang, Hsiu-Chien Chan, Rey-Ting Guo and Rong Xiao
Chemical Communications 2014 - vol. 50(Issue 58) pp:NaN7772-7772
Publication Date(Web):2014/05/01
DOI:10.1039/C4CC01752H
Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs' stereoselectivity.
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