Enantioselective Nitrilase from Pseudomonas putida: Cloning,Heterologous Expression,and Bioreactor Studies |
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Authors: | Anirban Banerjee Sachin Dubey Praveen Kaul Brajesh Barse Markus Piotrowski U. C. Banerjee |
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Affiliation: | Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, India. |
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Abstract: | 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. |
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Keywords: | Nitrilase cloning Heterologous expression Pseudomonas putida (R)-(− )-mandelic acid Bioreactor studies |
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