期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 20, 期 24, 页码 -出版社
MDPI
DOI: 10.3390/ijms20246224
关键词
metagenome-derived beta-glucosidase; directed evolution; site-directed mutagenesis; thermostability; butyl glucoside
资金
- National Natural Science Foundation of China [31760437]
- Natural Science Foundation of Guangxi Zhuang Autonomous Region of China [2018 GXNSFAA050090, 2017GXNSFBA198101]
- Guangxi Education Innovation Program for Postgraduates
- College Student Innovation-Venture Training Program of Guangxi University [YCSZ2012015]
- Study Abroad Program for Excellent Ph.D. Students of Guangxi Zhuang Autonomous Region
Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived beta-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (k(cat)/K-m of 561.72 mM(-1) s(-1)) toward rho-nitrophenyl-beta-d-glucopyranoside (rho NPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 degrees C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to beta-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel beta-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.
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