Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase

Enzyme and Microbial Technology
Ana ChenSong Li

Abstract

Pullulanase is a starch-debranching enzyme that is generally employed to efficiently break down starch for the production of high-glucose syrup. Acidic adaptation of pullulanases is of special interest. In this study, we conducted protein engineering to improve the acidic adaptation of Bacillus acidopullulyticus pullulanase (BaPul) and used a hydrogen-bond-based approach to identify promising residues that may change the deprotonation constants (pKa) of the catalytic residues. A total of 19 amino acids were selected for mutation according to the crystal structure of BaPul. The pH optimum of the L627R mutant shifted from 5.0 to 4.0, and its relative activity at pH 4.0 was 117% that of the wide-type enzyme. The improved efficacy of the L627R mutant at pH 4.0 was confirmed by kinetic parameters and pKa prediction. Moreover, the L627R mutant exhibited increased tolerance against acid-mediated denaturation, and its maximum d-glucose content (97.4%) was obtained after 40 h incubation, which is shorter by 10 h compared with the time required by the wide-type enzyme to produce a comparable amount of the monosaccharide. The L627R mutant may be suitable for industrial application because its shortened reaction time translates to reduced ...Continue Reading

Citations

Feb 7, 2021·World Journal of Microbiology & Biotechnology·Pei XuWen-Yong Lou
Jun 21, 2021·Biotechnology Advances·Wei XiaJing Wu
Dec 7, 2021·Critical Reviews in Biotechnology·Shu-Fang LiYu-Guo Zheng

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