title: Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart.
abstract: The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (Tm) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of Km and kcat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Bi-Lin
authors.initials: BL
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Meihong
authors.initials: M
authors.lastname: Dai
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Yuanhao
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Dongheng
authors.initials: D
authors.lastname: Meng
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Yasi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China tangb@whu.edu.cn.
authors.forename: Bing
authors.initials: B
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D012697:Serine Endopeptidases