Functional Characterization of a Novel Microbial Psychrophilic Lipase and Its Utilization in Stereo-Selective Biocatalysis

doi:10.13523/j.cb.20161006

China Biotechnology

2016, Vol. 36

Issue (10): 35-44 DOI: 10.13523/j.cb.20161006

Functional Characterization of a Novel Microbial Psychrophilic Lipase and Its Utilization in Stereo-Selective Biocatalysis

GONG Yan-hui¹, MA San-mei¹, ZHANG Yun^2,3, WANG Yong-fei¹, HU Yun-feng^2,3

1. Department of Biotechnology, Jinan University, Guangzhou 510632, China;
2. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
3. Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

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Abstract

From the genome of Pseudomonas antitumoralis HUP007, an esterase gene harboring 1041 bp and encoding a protein EstP8 of 377 amino acid was cloned. Esterase EstP8 was efficiently expressed in E. coli BL21(DE3) and further purified. Esterase EstP8 belongs to the IV family of lipases and contains conserved HGGG motif. The optimal substrate of esterase EstP8 was p-NPO, the optimal working temperature of esterase EstP8 was 50℃ and the optimal working pH of esterase EstP8 was 8.0. The hydrolysis activity of esterase EstP8 toward p-NPO was 105.19 U/mg, with V_max and K_m being 89.4 μmol/min and 1.144 mM, respectively. Esterase EstP8 remained high activity at pH7.0~8.0. The relative activity of esterase EstP8 was 41.78% at 4℃ and esterase EstP8 behaved very good thermo-stability at 10~40℃. EstP8 exhibited very good resistance to most metal ions tested. The addition of Cu²⁺, Mn²⁺ or Zn²⁺ of low concentrations could even stimulate the activity of EstP8. Organic solvents such as isooctane, methylbenzene, acetone and DMF could stimulate the activity of EstP8. Esterase EstP8 could generate chiral (R)-1-phenylethanol through kinetic resolution. The addition of organic solvents could well stimulate the stereo-selectivity and conversion during kinetic resolution. The e.e. and conversion of generated (R)-1-phenylethanol could reach 91% and 18%, respectively, in the presence of methylbenzene. The e.e. and conversion of generated (S)-styrallyl acetate could reach 98% and 60%, respectively, in the presence of DMSO. Psychrophilic esterase EstP8 possesses good potential in diverse industries such as stereo-selective biocatalysis.

Key words： Resistance to metal ions Psychrophilic esterase Kinetic resolution Resistance to organic solvents High enantio-selectivity

Received: 16 March 2016 Published: 25 October 2016

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	GONG Yan-hui
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GONG Yan-hui, MA San-mei, ZHANG Yun, WANG Yong-fei, HU Yun-feng. Functional Characterization of a Novel Microbial Psychrophilic Lipase and Its Utilization in Stereo-Selective Biocatalysis. China Biotechnology, 2016, 36(10): 35-44.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20161006 OR https://manu60.magtech.com.cn/biotech/Y2016/V36/I10/35

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