Cloning, expression, and characterization of a thermostable l‐arginase from Geobacillus thermodenitrificans NG80‐2 for l‐ornithine production. (24th June 2015)

• Record Type: Journal Article
• Authors: Huang, Kai; Mu, Wanmeng; Zhang, Tao; Jiang, Bo; Miao, Ming
• Is Part Of: Biotechnology and applied biochemistry. Volume 63:Number 3(2016:May/Jun.)
• Page Start: 391
• Page End: 397
• Publication Date: 2015-06-24
• Subjects: Geobacillus thermodenitrificans -- l‐arginase -- l‐ornithine -- l‐arginine -- gene expression -- biocatalysis; Biotechnology -- Periodicals; Biochemical engineering -- Periodicals; Biochemistry -- Periodicals; Biochemistry -- Periodicals; Genetic Techniques -- Periodicals; Microbiological Techniques -- Periodicals; 660.6
• DOI: 10.1002/bab.1385
• Languages: English
• ISSNs: 0885-4513

Abstract

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 producel ‐ornithine froml ‐arginine. In this article, thel ‐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 thel ‐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 thel ‐arginase activity evaluated by inactivation agents. The biosynthesis yield ofl ‐ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.