Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies.
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.
Banerjee
Anirban
A
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, India.
Dubey
Sachin
S
Kaul
Praveen
P
Barse
Brajesh
B
Piotrowski
Markus
M
Banerjee
U C
UC
D000097:Acetonitriles / Q000378:metabolism; D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism*; D019149:Bioreactors; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008333:Mandelic Acids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
D000097:Acetonitriles; D008333:Mandelic Acids; D011994:Recombinant Proteins; C045516:mandelonitrile; D000619:Aminohydrolases; C021867:nitrilase