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Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinis YC‐9 to improve its catalytic efficiency

Author:
Mao, Shurui, Gao, Peng, Lu, Zhaoxin, Lu, Fengxia, Zhang, Chong, Zhao, Haizhen, Bie, Xiaomei
Source:
Journal of the science of food and agriculture 2016 v.96 no.1 pp. 109-115
ISSN:
0022-5142
Subject:
Bacillus altitudinis, acidity, amino acids, beta-glucanase, catalytic activity, directed evolution, engineering, enzyme activity, mutants, pH, polymerase chain reaction, screening, sequence analysis, thermal stability
Abstract:
BACKGROUND: Error‐prone polymerase chain reaction (PCR) is frequently used in directed evolution of enzymes to modify their quality. In this study, error‐prone PCR was used to improve the catalytic efficiency of β‐1,3‐1,4‐glucanase from Bacillus altitudinis YC‐9. RESULTS: By screening, the mutant Glu‐3060 with higher activity was selected among 5000 transformants. After induction with isopropyl β‐d‐1‐thiogalactopyranoside (IPTG), the activity of the mutant Glu‐3060 reached 474.6 U mL⁻¹, resulting in a 48.6% increment of the parent enzyme activity. Research on the characterization of the mutated enzyme showed the optimal pH of the mutated enzyme to be 5.0, which is lower than the parent enzyme, but thermal stability was almost the same between them. Sequence analysis of the mutated enzyme revealed that three amino acids were changed compared with the parent enzyme, including K142N, Q203L and N214D. CONCLUSION: The three‐dimensional structure predicted by SWISS‐MODEL of the mutated enzyme Glu‐3060 showed that the substitution of three amino acids had an effect on the catalytic activity, stability and optimal pH of the enzyme, through changing the charge properties or electron density, forming secondary keys, the acidity of the amino acids and the side chain group. The sum effects of all the factors were increased activity of the mutated enzyme and decreased optimal pH, while the same thermostability was maintained, thereby increasing the suitability of the enzyme for industrial use. © 2014 Society of Chemical Industry
Agid:
4789528