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Cloning, expression, and characterization of a thermostable l ‐arginase from Geobacillus thermodenitrificans NG80‐2 for l ‐ornithine production
Author(s) -
Huang Kai,
Mu Wanmeng,
Zhang Tao,
Jiang Bo,
Miao Ming
Publication year - 2015
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1385
Subject(s) - arginase , ornithine , enzyme , biochemistry , enzyme kinetics , arginine , affinity chromatography , escherichia coli , cofactor , microbiology and biotechnology , specific activity , molecular mass , biology , size exclusion chromatography , biosynthesis , recombinant dna , chemistry , amino acid , active site , gene
Arginase ( l ‐arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l ‐ornithine from l ‐arginine. In this article, the l ‐arginase gene encoding the Geobacillus thermodenitrificans NG80‐2 was cloned and overexpressed in Escherichia coli . The specific activity of the purified enzyme was 138.3 U/mg. The molecular mass of the l ‐arginase was approximately 33.0 kDa as estimated by SDS‐PAGE and 192.0 kDa as determined by gel‐filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg 2+ , Cu 2+ , Ba 2+ , Ca 2+ , and Zn 2+ . Activity was optimal at pH 9.0 and 80 °C, and the protein was stable at 40 and 50 °C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis–Menten constant ( K m ) and catalytic efficiency ( k cat / K m ) were measured to be 171.9 mM and 3.8 mM −1 s −1 , respectively. Trp and His residues were directly involved in the l ‐arginase activity evaluated by inactivation agents. The biosynthesis yield of l ‐ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.