[Cloning, expression and purification of D-carbamoylase from Sinorhizobium morelens S-5].
A N-carbamoyl-D-amino acid amidohydrolase gene (hyuC) from Sinorhizobium morelens S-5 was cloned by LA PCR, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of D-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the recombinant enzyme was purified 16.1-fold to homogeneity with a yield of 21.2% by heat treatment and three steps of column chromatography. The results of gel filtration on Superdex 200 HR and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 38-kDa subunits. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and it was strictly D-specific. The enzyme showed broad substrate specificity, and exhibited high activity in the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine as substrate. The enzyme did not hydrolyze N-carbamoyl-beta-alanine. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. Enzyme activity was slightly improved by Ca2+ and Fe2+, and nearly not affected by metal chelators and sulfhydryl reagents. The enzyme showed high thermal and oxidative stability. These results show that the enzyme has great potential for industrial application.
Wu
Sheng
S
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Bejing 100080, China. wush@sun.im.ac.cn
Wang
Jian-jun
JJ
Yang
Liu
L
Sun
Wan-ru
WR
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D020579:Sinorhizobium / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D001426:Bacterial Proteins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase