title: Improved thermostability of esterase from Aspergillus fumigatus by site-directed mutagenesis.
abstract: A 1.020-bp esterase gene, estQ, encoding for a protein of 339 amino acids, was cloned from Aspergillus fumigatus and expressed in E. coli. EstQ exhibited the optimal activity around 40 °C and pH 9.0. In order to obtain more thermostable esterases, three mutants (A134T, V160T, A134T-V160T) were constructed by site-directed mutagenesis and also characterized for further research. Compared to A134T and V160T displaying their optimum activity at 40 °C, A134T-V160T exhibited a 5 °C higher optimal temperature and a longer half-life more than 24 times than that of WT at 50 °C. All the mutants displayed favorable effects on thermostability and retained 53-76% activity after pre-incubation for 30 min at 45 °C, about 20-40% higher than that of the WT. With an increase in Km of the three mutants, a decrease in catalytic efficiency in kcat/Km was observed in mutant V160T and A134T-V160T against p-nitrophenyl butyrate. Homology models of WT and A134T-V160T were built to understand the structure-function relationship. The analysis results showed that the improved thermostability may be due to the favorable interaction and additional hydrogen bonds formed in the mutants by substitution of hydrophobic residues with hydrophilic residues. This study provide useful theoretical reference for enzyme evolution in vitro.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
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, PR China.
authors.forename: Shiyu
authors.initials: S
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. Electronic address: lzd@mail.hzau.edu.cn.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020240:Apraxia, Ideomotor; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005800:Genes, Fungal; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D004950:Esterases
keywords: Esterase; Half-life; Site-directed mutagenesis; Thermostability