title: Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa.
abstract: The lipase from Malassezia globosa (SMG1) has specific activity on mono- and diacylglycerol but not on triacylglycerol. The structural analysis of SMG1 structure shows that two bulky aromatic residues, W116 and W229, lie at the entrance of the active site. To study the functions of these two residues in the substrate recognition and the catalytic reaction, they were mutated to a series of amino acids. Subsequently, biochemical properties of these mutants were investigated. Although the activities decrease, W229L and W116A show a significant shift in substrate preference. W229L has an increased preference for short-chain substrates whereas W116A has preference for long-chain substrates. Besides, the half-lives of W116A and W116H at 45 °C are 346.6 min and 115.5 min respectively, which improve significantly compared to that of native enzyme. Moreover, the optimum substrate of W116A, W116F and W229F mutants shifted from p-nitrophenyl caprylate to p-nitrophenyl myristate. These findings not only shed light onto the lipase structure/function relationship but also lay the framework for the potential industrial applications.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China.
authors.forename: Chongliang
authors.initials: C
authors.lastname: Gao
authors.affiliation: College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, PR China.
authors.forename: Dongming
authors.initials: D
authors.lastname: Lan
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China.
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, PR China.
authors.forename: Houjin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.affiliation: College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, PR China. Electronic address: yonghw@scut.edu.cn.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D008290:Malassezia / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013379:Substrate Specificity; D014280:Triglycerides / Q000737:chemistry*
chemical_list: D014280:Triglycerides; D008049:Lipase
keywords: CDocker; Catalytic pocket; Lipase; Site-directed mutagenesis; Substrate selectivity; Thermostability