Systems Microbiology and Biomanufacturing
·
2022
·
IF 0
Zhu Jiufeng,Bai Yajun,Fan Tai-Ping,Zheng Xiaohui,Cai Yujie
Ethyl ( R )-4-chloro-3-hydroxybutyrate (( R )-CHBE), as a chiral intermediate, is widely used in the synthesis of various chiral drugs. In this study, we screened two aldo–keto reductases (LP-AKRs) from the probiotic Lactobacillus plantarum DSM20174, both with a molecular weight of approximately 31 kDa. Both enzymes could reduce 4-chloroacetoacetic acid ethyl ester (COBE) to produce ( R )-CHBE with an enantioselectivity value of 99%. When determining the kinetic parameter, the K m , K cat , and V max of LP-AKR5 and LP-AKR9 were 9.5 mM, 7.6 U/mg, 3.96 s −1 and 8.7 mM, 8.59 U/mg, 4.47 s −1 , respectively. Both LP-AKR5 and LP-AKR9 had an optimal reaction pH of 6 and could maintain a high level of stability at pH 6, allowing them to perform well in an acidic environment. LP-AKR5 and LP-AKR9 had optimal reaction temperatures of 30 °C and 40 °C, respectively. Metal ions had minimal influence on LP-AKR5 and LP-AKR9 enzyme activities. This series of enzymatic properties showed that LP-AKR5 and LP-AKR9 mined from Lactobacillus plantarum DSM20174 could asymmetrically catalyze the synthesis of ( R )-CHBE under weakly acidic circumstances, which could maintain product stability and provide a good foundation for industrial production.
研究领域:
生物化学
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分子生物学
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工业微生物学
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DOI
PROTEIN EXPRESSION AND PURIFICATION
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2015
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IF 1.695
Jian-yong Zheng,Jian Wang,Sha-sha Zhou,Xiao-jun Li,Xiang-xian Ying,Zhao Wang
Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert ( S )-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to ( S )-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining ( R )-CHBE and formed ( S )-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac -CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual ( R )-CHBE and the formed ( S )-HL had high optical purities ( e.e. s > 99.9%, e.e. p > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.
研究领域:
生物化学
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分子生物学
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工业酶学
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产品关联
DOI
PubMed
高影响因子
CATALYSIS LETTERS
·
2019
·Vol. 149
·期 11
·2973-2983
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IF 2.372
Luo Wei,Du Hui-Jun,Bonku Emmanuel Mintah,Hou Ya-Li,Li Ling-Ling,Wang Xiao-Qiang,Yang Zhong-Hua
Abstract Enantiopure alcohols have received much attention due to their widespread use as pharmaceutical intermediates. In the asymmetric biosynthesis of enantiopure alcohols, the excellent performance of carbonyl reductase makes it be the best choice as the biocatalysts. In this work, an alkali-tolerant carbonyl reductase ( Bs CR, encoded by yueD ) from Bacillus subtilis (strain 168) was obtained through gene mining, and successfully heterologously expressed in Escherichia coli with pET-32a. Bs CR showed excellent alkali resistance and even can keep more than 70% of its peak activity after incubation in Tris–HCl buffer at pH 9.0 for 40 h. The Michaelis constants and maximal velocity of the Bs CR to NADPH (A) and ethyl 4-chloroacetoacetate (B) are \(K_{m}^{A}\) = 5.390 × 10 −2 mmol/L, \(K_{m}^{B}\) = 1.855 mmol/L, and \(V_{max}\) = 147.3 μmol·min −1 ·mg −1 , respectively. Applying the E. coli BL21(DE3)/pET-32a-yueD to catalyze asymmetric reduction of ethyl 4-chloroacetoacetate and acetophenone, the yield of S -CHBE reached 89.9% and S -1-phenyl ethanol reached 66.7%, and e.e. of both products reached more than 99%. This work provides a novel CR for asymmetric reduction. Graphic Abstract A carbonyl reductase ( Bs CR) and its gene were identified through gene mining, and overexpressed in Escherichia coli BL21(DE3) for whole-cell biocatalytic asymmetric reduction
研究领域:
生物技术
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生物化学
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药物科学
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酶学
✓ 已验证
DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2016
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IF 4.917
Yu-Cai He,Dan-Ping Zhang,Jun-Hua Di,Yin-Qi Wu,Zhi-Cheng Tao,Feng Liu,Zhi-Jun Zhang,Gang-Gang Chong,Yun Ding,Cui-Luan Ma
In this study, sugarcane bagasse (SB) was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated SBs, it was found that all these three concentrated hydrolyzates could be used for the asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl ( S )-4-chloro-3-hydroxybutanoate [( S )-CHBE]. Compared with glucose, arabinose and cellobiose couldn’t promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for the effective biosynthesis of ( S )-CHBE (∼500 g/L; 98.0% yield) from 3000 COBE by whole cells of Escherichia coli CCZU-K14 in the presence of β-CD (0.4 mol β-CD/mol COBE), l -glutamine (200 mM) and glycine (500 mM). In conclusion, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate ( S )-CHBE.
研究领域:
生物技术
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生化工程
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有机化学
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微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）