Cross-linked enzyme aggregates of recombinant Pseudomonas putida nitrilase for enantioselective nitrile hydrolysis.
The cross-linked enzyme aggregate (CLEA) method is used for the dual purpose of combining both the purification and immobilization of enzyme in one step. The present work involved the preparation of a carrier-free, highly active reusable biocatalyst (nitrilase) which encounters least mass-transfer limitations with higher thermal and storage stability. The effect of type of aggregating agent, its concentration as well as that of cross-linking agent was studied. Nitrilase aggregates were prepared using ammonium sulphate (35%) precipitation followed by cross-linking with glutaraldehyde (125 mM) which rendered 70% activity retention. The various cross-linking parameters were optimized in order to increase the activity retention. Stability in terms of temperature, reusability and leaching were also examined. The CLEA preparation showed residual nitrilase activity on repeated use. A highly stable CLEA of nitrilase was finally prepared with maximum activity recovery.
Kumar
Sandeep
S
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, SAS Nagar, 160 062 Punjab, India.
Mohan
Utpal
U
Kamble
Ashwini L
AL
Pawar
Sandip
S
Banerjee
Uttam C
UC
D000619:Aminohydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011995:Recombination, Genetic; D013237:Stereoisomerism
D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase