title: Characterization and directed evolution of BliGO, a novel glycine oxidase from Bacillus licheniformis.
abstract: Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40°C. Interestingly, BliGO retained 60% of the maximum activity at 0°C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and k(cat)/K(m)) of BliGO were 11.22 mM, 0.08 s(-1), and 0.01 mM(-1) s(-1), respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM(-1) s(-1)) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: wgb@mail.hzau.edu.cn.
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production and Construction Crops, College of Life Science, Tarim University, Alar 843300, China. Electronic address: zhang63lyly@sina.com.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000069976:Bacillus licheniformis / Q000201:enzymology* / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase
keywords: DNA shuffling; Directed evolution; Glycine oxidase; Glyphosate