Enantioselective Nitrilase from Pseudomonas putida: Cloning, Heterologous Expression, and Bioreactor Studies
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Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.
KeywordsNitrilase cloning Heterologous expression Pseudomonas putida (R)-(−)-mandelic acid Bioreactor studies
Anirban Banerjee gratefully acknowledges financial assistance provided by CSIR Govt. of India, and DAAD Fellowship, Sachin Dubey and Praveen Kaul gratefully acknowledge financial assistance provided by DBT and CSIR, Govt. of India. This is NIPER communication number 413.
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