Abstract
Objectives
To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing.
Results
A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg−1 and 189 s−1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml−1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively.
Conclusions
The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.
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Acknowledgments
The authors appreciated Prof. Wilfred J Mitchell (School of Life Sciences, Heriot-Watt University) for providing laboratory space and equipment. The authors are also grateful to Mrs. Vicky Goodfellow (International Centre for Brewing and Distilling, Heriot-Watt University) for her help in congress mash preparation and analysis. This study was financially supported by the National High Technology Research and Development Program 863 (No.2013AA102106), National Science Foundation (Nos. 31271919, 31571942 & 31301539), and the Fundamental Research Funds for the Central Universities (JUSRP51504, JUSRP51402A & JUDCF13008), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Program of Introducing Talents of Discipline to Universities (No.111-2-06).
Supporting information
Supplementary Table 1—Comparison of secondary structure between the wild-type BglT and rE-BglTO using Dichroweb online software.
Supplementary Fig. 1—Amino acid sequences of the BglT gene and rE-BglTO gene.
Supplementary Fig. 2—SDS-PAGE analysis of purified the wild-type and recombinant 1,3-1,4-β-glucanases.
Supplementary Fig. 3—Comparison of electrostatic surface potential of the wild-type BglT and rE-BglTO
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Niu, C., Zhu, L., Hill, A. et al. Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry. Biotechnol Lett 39, 113–122 (2017). https://doi.org/10.1007/s10529-016-2212-2
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DOI: https://doi.org/10.1007/s10529-016-2212-2