Thermostable D-carbamoylase from Sinorhizobium morelens S-5: purification, characterization and gene expression in Escherichia coli.
A d-carbamoylase from Sinorhizobium morelens S-5 was purified and characterized. The enzyme was purified 189-fold to homogeneity with a yield of 19.1% by aqueous two-phase extraction and two steps of column chromatography. The enzyme is a homotetramer with a native molecular mass of 150 kDa and a subunit relative molecular mass of 38 kDa. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. The enzyme showed high thermal and oxidative stability. It was found to have a K(m) of 3.76 mM and a V(max) of 383 U/mg for N-carbamoyl-d-p-hydroxyphenylglycine. The hyuC gene coding for this enzyme was cloned, 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 product was purified to homogeneity from the recombinant. Our results show that the enzyme has great potential for industrial application.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, China.
Sheng
S
Wu
Yanbin
Y
Liu
Guogang
G
Zhao
Jianjun
J
Wang
Wanru
W
Sun
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D020579:Sinorhizobium / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D021122:Protein Subunits; D011994:Recombinant Proteins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase