title: Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis.
abstract: The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the Km values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi, Jiangsu Province, China.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi, Jiangsu Province, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Cheng
authors.affiliation: Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong Province, China.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Ai
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi, Jiangsu Province, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D005964:Glucosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007534:Isomaltose / Q000031:analogs & derivatives / Q000096:biosynthesis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012705:Serratia / Q000201:enzymology*; D040681:Structural Homology, Protein; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D007534:Isomaltose; D005964:Glucosyltransferases; C116853:sucrose isomerase; C008189:isomaltulose