Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir

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• 作者：Xiu-Juan He ; Shao-Yun Chen ; Jian-Ping Wu…
• 关键词：Lactobacillus kefir alcohol dehydrogenase ; tert ; Butyl (S) ; 6 ; chloro ; 5 ; hydroxy ; 3 ; oxohexanoate ; Statins ; Engineered enzyme ; Fed ; batch strategy
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：November 2015
• 年：2015
• 卷：99
• 期：21
• 页码：8963-8975
• 全文大小：1,131 KB
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• 作者单位：Xiu-Juan He (1)
  Shao-Yun Chen (2)
  Jian-Ping Wu (1)
  Li-Rong Yang (1)
  Gang Xu (1)
  
  1. Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, Hangzhou, 310027, People’s Republic of China
  2. College of Life Science, Zhejiang Chinese Medical University, 548# Binwen Road, Hangzhou, 310053, People’s Republic of China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) is a valuable chiral synthon, which is used for the synthesis of the cholesterol-lowering drugs atorvastatin and rosuvastatin. To date, only the alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefir (LkADH) have demonstrated catalytic activity toward the asymmetric reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) to (S)-CHOH. Herein, a tetrad mutant of LkADH (LkTADH), A94T/F147L/L199H/A202L, was screened to be more efficient in this bioreduction process, exhibiting a 3.7- and 42-fold improvement in specific activity toward CDOH (1.27 U/mg) over LbADH (0.34 U/mg) and wild-type LkADH (0.03 U/mg), respectively. The molecular basis for the improved catalytic activity of LkTADH toward CDOH was investigated using homology modeling and docking analysis. Two major issues had a significant impact on the biocatalytic efficiency of this process, including (i) the poor aqueous stability of the substrate and (ii) partial substrate inhibition. A fed-batch strategy was successfully developed to address these issues and maintain a suitably low substrate concentration throughout the entire process. Several other parameters were also optimized, including the pH, temperature, NADP+ concentration and cell loading. A final CDOH concentration of 427 mM (100 g/L) gave (S)-CHOH in 94 % yield and 99.5 % e.e. after a reaction time of 38 h with whole cells expressing LkTADH. The space–time yield and turnover number of NADP+ in this process were 10.6 mmol/L/h and 16,060 mol/mol, respectively, which were the highest values ever reported. This new approach therefore represents a promising alternative for the efficient synthesis of (S)-CHOH.