Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 176, Issue 6, Pages 1736-1745Publisher
HUMANA PRESS INC
DOI: 10.1007/s12010-015-1675-2
Keywords
Pullulanase; Site-saturated mutagenesis; Thermostability; Optimum temperature.; Catalytic efficiency
Funding
- National Key Basic Research and Development Program of China (973 Program) [2011CB710800]
- National High Technology Research and Development Program of China (863 Program) [2012AA022207]
- National Natural Science Foundation of China (NSFC) [21376107, 21336009]
- 111 Project [111-2-06]
- High-end Foreign Experts Recruitment Program [GDW20133200113]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- Jiangsu province Collaborative Innovation Center for Advanced Industrial Fermentation industry development program
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Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 degrees C, and correspondingly the half lives of these mutants at 55 degrees C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of V-max and k(cat)/K-m, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently.
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