Co-reporter:Yongxian Fan, Yuele Lu, Liwei Zhang, Xiaolong Chen, Yinchu Shen
Biocatalysis and Agricultural Biotechnology 2017 Volume 11(Volume 11) pp:
Publication Date(Web):1 July 2017
DOI:10.1016/j.bcab.2017.08.004
Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, their highly-effective industrial application is often limited by the regeneration of cofactor NADPH. In this study, G6PDH from Rhizopus oryzae was expressed to enhance the regeneration of NADPH in the industrial strain, R. nigricans. The results showed the fermentation time of the engineered strain was shortened to 52 h when the hydroxylation yields reached the maximum, which was 4 h ahead compared to that of the original strain. 95.4%-higher-level NADPH could be generated and the hydroxylation yield increased by 15.6% in an engineered strain after 52-h fermentation compared to those in the original strain after 56-h fermentation. The results indicated that the strategy of enhancing NADPH regeneration by expressing G6PDH was simple and effective on improving the P450-monooxygenase-dependent hydroxylation ability in filamentous fungi.
Co-reporter:Yuele Lu;Yihua Shao;Songjun Dai;Jinling Diao;Xiaolong Chen
Chirality 2016 Volume 28( Issue 5) pp:394-398
Publication Date(Web):
DOI:10.1002/chir.22589
Abstract
Benalaxyl is widely applied as a fungicide during grape planting processing. In this experiment, the stereoselective behavior of benalaxyl was studied during the grape growth and wine-making process. A simple method based on high-performance liquid chromatography (HPLC) equipped with a chiral column and UV detector was established to separate and determine the enantiomers of benalaxyl. Stereoselective degradation of the two enantiomers of benalaxyl was found in grapes. The degradation of both enantiomers followed pseudofirst-order kinetics, and the degradation rate of R-(−)-benalaxyl was faster than S-(+)-benalaxyl. The half-life of R-(−)-benalaxyl was 27 h, while the half-life of S-(+)-benalaxyl was 31 h. The enantiomer fraction value decreased from 0.50 to 0.34 and finally only S-(+)-benalaxyl could be detected. In the fermentation process, both enantiomers of benalaxyl were hardly degraded, and no configuration interconversion was observed. Meanwhile, both enantiomers of benalaxyl showed little influence on the growth of the yeast, consumption of carbon sources, or production of alcohol. The result of this study might provide more sufficient data for the evaluation of food safety and potential risk. Chirality 28:394–398, 2016. © 2016 Wiley Periodicals, Inc.
Co-reporter:Yuele Lu;Dong Zhang;Yahui Liao;Jinling Diao;Xiaolong Chen
Chirality 2016 Volume 28( Issue 1) pp:78-84
Publication Date(Web):
DOI:10.1002/chir.22545
Abstract
Chiral pesticides are now receiving more and more attention in the food-making process. This experiment studied the enantioselective behavior of diclofop-methyl (DM) and its main metabolite, diclofop (DC), during the soy sauce brewing process. Two kinds of commonly used strains, Aspergillus oryzae and Saccharomyces rouxii, were investigated. However, they showed a different degradation ability to the enantiomers of DM and DC. It was observed that (–)-(S)-DM was degraded much faster than (+)-(R)-DM by Saccharomyces rouxii, while no stereoselective degradation was found by Aspergillus oryzae. DC represented a relatively long residue period in this fermentation process and both strains showed a weak degradation ability to DC, especially Saccharomyces rouxii. There was little DC detected in the final product, while most of the DC residues persisted in the lees, which were usually used as animal feeds or discarded into the environment directly as waste. Therefore, more attention should be paid to the soy sauce brewing process concerning pesticide residues both in the final product and byproducts. Chirality 28:78–84, 2016. © 2015 Wiley Periodicals, Inc.
Co-reporter:Rui Guo, Yong-Xian Fan, Xiao-Long Chen, and Yin-Chu Shen
Journal of Agricultural and Food Chemistry 2013 Volume 61(Issue 1) pp:157-166
Publication Date(Web):December 5, 2012
DOI:10.1021/jf303616s
The d-threo form of p-methylsulfonylphenyl serine ethyl ester (MPSE) is a key intermediate for the synthesis of florfenicol. In this study, chiral resolution of dl-threo-p-MPSE with lipases was investigated. Among a series of lipases, Novzyme 435 was the best to resolve dl-threo-p-MPSE with the conversion rate of 36.83% and ee value of 35.13%. To improve the conversion rate and ee value, a number of byproducts were identified and characterized using reverse-phase HPLC, normal-phase HPLC, 1H NMR, and LC-MS when threo-p-MPSE was hydrolyzed by lipases in organic medium. Mechanisms of generating main byproducts are proposed, and a suppressing method is provided. The results showed that byproduct p-methylsulfonyl benzaldehyde serves as the key intermediate during the whole side reaction process. It was also observed that threo-p-MPSE with a proper hydrolytic velocity served as a driving force to generate p-methylsulfonyl benzaldehyde and accelerated the side reactions. Finally, a feasible approach to suppress side reactions in enzymatic catalysis is offered. The conversion rate and ee value were greatly improved by 69.29 and 46.26%, respectively, using Zn2+ compared to those without Zn2+.
Co-reporter:Xiaolong Chen, Xiaohui Zhu, Yicheng Ding and Yinchu Shen
The Journal of Antibiotics 2011 64(8) pp:563-569
Publication Date(Web):July 20, 2011
DOI:10.1038/ja.2011.55
The potential of tautomycin to control oilseed rape stem rot was investigated in this paper. Tautomycin produced by Streptomyces spiroverticillatus strongly inhibited Sclerotinia sclerotiorum, which causes oilseed rape stem rot. Tautomycin showed great inhibition of the mycelial growth of S. sclerotiorum on potato dextrose agar (PDA) plates. The values of EC50 and MIC were 3.26 × 10−4 mM and 6.52 × 10−4 mM, respectively. Tautomycin treatment also resulted in morphological abnormalities of S. sclerotiorum such as hyphal swellings and abnormally branched shapes, which were observed microscopically. Sclerotia of S. sclerotiorum soaked in the tautomycin solution for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA plates was completely inhibited when the concentration of tautomycin reached 6.52 × 10−4 mM. Tautomycin-treated oilseed rape leaves were found to have a low incidence of leaf blight caused by S. sclerotiorum. The activity of the protein phosphatase (PP) in S. sclerotiorum decreased by 41.6% and 52.6% when treated with 3.30 × 10−4 mM and 6.52 × 10−4 mM tautomycin, respectively. Cellular constituents also leaked from S. sclerotiorum cells incubated with tautomycin. The results suggest that the antimicrobial activity of tautomycin is due to the inhibition of the PP and then a change of membrane permeability. This paper also investigated related compounds that possess either a maleic anhydride or maleic acid moiety. Results showed 2,3-dimethylmaleic anhydride, diphenylmaleic anhydride and dimethyl maleate demonstrated significant activity against S. sclerotiorum. The values of EC50 for these three compounds were 0.31 mM, 0.15 mM and 3.99 mM, respectively. The MIC values obtained for these compounds were 1.11 mM, 0.56 mM and 9.58 mM, respectively.
Co-reporter:Xiao-Long Chen;Yu-Hua Xu;Yu-Guo Zheng
Biotechnology and Bioprocess Engineering 2010 Volume 15( Issue 6) pp:969-974
Publication Date(Web):2010 December
DOI:10.1007/s12257-010-0087-y
Optimization of the feeding process for tautomycin production by Streptomyces spiroverticillatus was performed using glucose and/or maleic anhydride. The feeding of glucose was based on the reducing sugar content (lower than 8 g/L) at a cultivation time of 40 h. After addition of 2% (w/v) glucose, the biomass increased from 21 to 28 g/L, and that of tautomycin from 572.06 to 837.6 mg/L. Moreover, 1723.1 mg/L of tautomycin (increased by 201.21%) was obtained by feeding 0.2% (w/v) maleic anhydride solution at a pH between 4 and 7 in the broth. For the experiments in the 15 L fermentor, tautomycin content reached its highest level (1714.7 mg/L), which was 199.7% higher than that of control by feeding both glucose and maleic anhydride.
Co-reporter:Xiaolong Chen, Xinrong Luo, Feifei Cao, Tingheng Zhu, Yongxian Fan, Xiaoqing Jia, Yinchu Shen
Enzyme and Microbial Technology (November 2014) Volume 66() pp:28-34
Publication Date(Web):1 November 2014
DOI:10.1016/j.enzmictec.2014.08.002
•The pCB1004-PgpdA was first used for cloning and expressing the CPR gene.•CPR gene was first strengthened in R. nigericans.•The strengthened expression of CPR gene can make great economic sense in steroid production.The hydroxylations of the steroid skeleton structure are catalyzed by a family of enzymes, the cytochromes P450 (CYPs). In this study, the pCB1004-PgpdA plasmid was used for cloning the cytochrome P450 reductase (CPR) gene from Rhizopus oryzae into Rhizopus nigericans to strengthen the expression of CPR gene in R. nigericans with REMI (Restriction Enzyme Mediate Integration) mediated protoplast transformation. The conditions for the protoplast production of R. nigericans were optimized as follows: 75 μg/mL yatalase, 50 μg/mL lywallzyme, fungus age of 12 h, digestion time of 3 h and digestion temperature of 30 °C. REMI mediated protoplast transformation with plasmid pCB1004-PgpdA into R. nigericans was performed to construct the transformants. More than 30 transformants were successfully selected from the hygromycin B-resistant plates and 6 transformants had the abilities to improve the biotransformation of 16α, 17-epoxyprogesterone. The highest biotransformation rate of the transformants was 65.38%, which was 7.06% higher than that of the original strain.
