title: Cloning, expression, and characterization of a thermostable l-arginase from Geobacillus thermodenitrificans NG80-2 for l-ornithine production.
abstract: 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 (Km ) and catalytic efficiency (kcat /Km ) 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%.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Kai
authors.initials: K
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Ming
authors.initials: M
authors.lastname: Miao
mesh_terms: D000595:Amino Acid Sequence; D001119:Arginase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005240:Feasibility Studies; D015870:Gene Expression; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009952:Ornithine / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D008670:Metals; D009952:Ornithine; D001119:Arginase
keywords: Geobacillus thermodenitrificans; biocatalysis; gene expression; l-arginase; l-arginine; l-ornithine