title: Enhancement of the thermostability of Streptomyces kathirae SC-1 tyrosinase by rational design and empirical mutation.
abstract: This study aimed to improve the thermostability of a newly cloned tyrosinase from Streptomyces kathirae SC-1. The POPMuSiC algorithm was applied to predict the folding free energy change (ΔDG) of amino acid substitution. Site-directed mutagenesis was used to construct mutants (Q7K, G234P, and Q7K/G234P), and the mutant, and wild-type enzymes were expressed in Escherichia coli (DE3). Compared to the wild-type tyrosinase, all three mutant enzymes showed improved thermal properties. The mutant with combined substitution (Q7K/G234P) showed the most pronounced shifts in temperature optima, about 10 °C upward, and the half-life for thermal inactivation at 60 °C, and melting temperatures were increased by 3 times and approximately 10 °C, respectively. Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design strategies in this study may also provide further insight into the thermostability of other industrial enzymes and suggest further potential industrial applications.
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Guo
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China. Electronic address: raozhm@jiangnan.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
authors.forename: Zaiwei
authors.initials: Z
authors.lastname: Man
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China; Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Shang-Tian
authors.initials: ST
authors.lastname: Yang
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase
keywords: POPMuSiC; Site-directed mutagenesis; Thermostability; Tyrosinase