In vitro and in silico characterization of a novel glutamate carboxypeptidase from Cohnella sp. A01

Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a ther-mophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioin-formatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 degrees C and 7.2, respectively. Fluorescence spectroscopy data elucidated the sec-ondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, Km and specific activity values were calculated as 0.108 mM and 687 mmol/min/mg, respectively. The enzyme activity was strongly inhibited when EDTA sequestered zinc ions. The half-life of this enzyme at 30 degrees C was 2012 min. Regarding the ability of rCP302 to degrade folic acid, and its long half-life at 37 degrees C, the normal temperature of many mammals, this enzyme can be introduced for further study for use in the pharmaceutical industry.(c) 2022 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.