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KMID : 1094720150200030473
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Biotechnology and Bioprocess Engineering
2015 Volume.20 No. 3 p.473 ~ p.487
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Discovery and expression of a Pseudomonas sp. esterase as a novel biocatalyst for the efficient biosynthesis of a chiral intermediate of pregabalin
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Xu Fangxin
Chen Shaoyun
Xu Gang
Wu Jianping
Yang Lirong
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Abstract
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The enantioselective biocatalytic hydrolysis of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (rac-cyanodiester, rac-CNDE) to obtain a key (S)-intermediate is crucial for the synthesis of Pregabalin. The gene estZF172 from Pseudomonas CGMCC No. 4184, which encoded a novel esterase EstZF172 with excellent stereoselective catalysis capacity of (R)-CNDE (E = 95), was obtained from genomic library construction through activity screening. EstZF172 was identified to be a new member of the bacterial esterase/lipase Family VIII by sequence alignment, phylogenetic tree analysis and homology model analysis, with preference toward shortchain p-nitrophenyl esters. The esterase was functionally expressed in E. coli BL21(DE3), exhibiting a 120-fold improvement in catalytic activity (4027.5 U/L) over the wild strain (33.43 U/L) toward CNDE. For the chiral hydrolysis of rac-CNDE catalyzed by recombinant cells, the optimum operating temperature and pH were determined to be 35°C and 8.5, respectively, based on the biochemical characterization of the purified EstZF172. Finally, the yield and ee of (S)-CNDE reached 47.7% and > 99.5% after reaction for 7 h with a substrate loading of 127.5 g/L (500 mM). The results suggested that EstZF172 is a potential biocatalyst for the synthesis of an important chiral intermediate of Pregabalin.
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KEYWORD
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Pseudomonas sp. esterase, enantioselective hydrolysis, classification, biochemical characterization, Pregabalin
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