Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

PLoS One. 2016 Feb 17;11(2):e0149208. doi: 10.1371/journal.pone.0149208. eCollection 2016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Escherichia coli
  • Glucosyltransferases / metabolism*
  • Hydrogen-Ion Concentration
  • Isomaltose / analogs & derivatives
  • Isomaltose / biosynthesis
  • Kinetics
  • Mutagenesis, Site-Directed*
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, Protein
  • Serratia / enzymology*
  • Structural Homology, Protein
  • Temperature

Substances

  • Mutant Proteins
  • Recombinant Proteins
  • Isomaltose
  • Glucosyltransferases
  • sucrose isomerase
  • isomaltulose

Grants and funding

This work was funded by grants from the National Science Fund for Distinguished Young Scholars (http://www.nsfc.gov.cn/publish/portal1/ #31425020), the National Natural Science Foundation of China (http://www.nsfc.gov.cn/publish/portal1/ #31271813 and #31401636), the project of outstanding scientific and technological innovation group of Jiangsu Province (http://www.jskjjh.gov.cn/13kjskj2/), the Natural Science Foundation of Jiangsu Province (http://www.jskjjh.gov.cn/13kjskj2/ #BK20140142), the 111 Project (http://www.dost.moe.edu.cn No. 111-2-06), the China Postdoctoral Science Foundation Funded Project (2015M580390), and the Production and Research Prospective Joint Research Project of Jiangsu Province (BY2015019-18). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.