Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study.
Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.
Singh
Renu
R
Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, 160 062, India.
Banerjee
Anirban
A
Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, 160 062, India.
Kaul
Praveen
P
Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, 160 062, India.
Barse
Brajesh
B
Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, 160 062, India.
Banerjee
U C
UC
Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, Punjab, 160 062, India. ucbanerjee@niper.ac.in.
D042481:Alcaligenes faecalis / Q000201:enzymology* / Q000302:isolation & purification*; D000619:Aminohydrolases / Q000096:biosynthesis* / Q000302:isolation & purification*; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002458:Cell Fractionation / Q000379:methods*; D007536:Isomerism; D044085:Microfluidics / Q000379:methods*; D013010:Sonication
D000619:Aminohydrolases; C021867:nitrilase