Enhanced catalytic efficiency of Bacillus amyloliquefaciens SS35 endoglucanase by ultraviolet directed evolution and mutation analysis

摘要

Bacillus amyloliquefaciens SS35 was subjected to ultraviolet irradiation to improve the enzymatic hydrolysis of lignocellulosic biomass. The resulting mutant, UV2, produced endoglucanase, carboxymethyl cellulase, CMCase-UV2 with 1.6-4.1-fold higher activity against cellulosic substrates than the wild type, CMCase-WT. CMCase-UV2 exhibited wider pH stability in the acidic range than CMCase-WT. The TLC analysis showed that the hydrolysis of CMC-Na and beta-glucan by CMCase-UV2 produced glucose along with cello-oligosaccharides and cellobiose in 45 min, whereas, CMCase-WT produced, only cellooligosaccharides and cellobiose in 120 min by endolytic mode of action. The hydrolysis of pretreated Pennisetum purpureum by CMCase-UV2 gave total reducing sugar yield 154.2 mg/g pretreated biomass in 48 h, which was 1.8-fold higher than CMCase-WT. CMCase-UV2 was the promising endoglucanase which improves the saccharification of lignocellulosic biomass therefore, it will improve the efficiency of the process, lignocellulose-based biorefineries for bioethanol production. The gene encoding cellulase was amplified from wild-type and UV2 strains using degenerate primers designed from phylogenetically related spp. Bacillus amyloliquefaciens KHG19 for family 5 glycoside hydrolase. Sequences analysis of genes from wild-type and UV2 strains showed the mutation, D233G. These results will provide information for protein engineering in designing mutant of endoglucanase for improved catalytic efficiency and pH stability. (C) 2019 Elsevier Ltd. All rights reserved.