title: Trimeric l-N-carbamoylase from newly isolated Brevibacillus reuszeri HSN1: a potential biocatalyst for production of l-α-amino acids.
abstract: l-N-carbamoylase was isolated from Brevibacillus reuszeri HSN1 and purified to homogeneity in three steps, which is a reasonably short protocol for native l-N-carbamoylase. The enzyme purification protocol resulted in ≈60-fold purification of l-N-carbamoylase with specific activity of 145 µmol/Min/mg. The subunit and native molecular mass were found to be 44.3 and 132 kDa, respectively. Temperature and pH optima were determined as 50°C and 8.5, respectively. The enzyme had retained ≈86% activity at 50°C when incubated for 60 Min and the half-life was determined as 180 Min at 50°C. N-carbamoyl-l-methionine (l-N-CMet) was found to be a preferred substrate with Km and Vmax values of ≈13.5 mM and ≈103 µmol/Min/mg, respectively. The broad substrate specificity with derivatives of N-carbamoyl amino acids is advantageous to be a better biocatalyst for production of corresponding l-α-amino acids. The enzyme activity was enhanced by 73% in the presence of 0.8 mM Mn(2+) ion during the biotransformation. In the batch experiment, ≈97% conversion of 5.0% l-N-CMet into enantiomerically pure l-methionine was achieved in 10 H when carried out at pH 8.0, 45°C, and 15% wet (w/v) cell loading, under controlled conditions. The overall merits of this enzyme show promise as a potential biocatalyst for l-α-amino acid production.
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India. hemraj@imtech.res.in
authors.forename: Hemraj S
authors.initials: HS
authors.lastname: Nandanwar
authors.forename: Rakesh M
authors.initials: RM
authors.lastname: Vohra
authors.forename: Gurinder S
authors.initials: GS
authors.lastname: Hoondal
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000596:Amino Acids / Q000096:biosynthesis*; D055162:Biocatalysis; D001704:Biopolymers / Q000378:metabolism*; D058675:Brevibacillus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001704:Biopolymers; D000581:Amidohydrolases; C104561:N-carbamoyl-L-amino-acid hydrolase