High-efficiency expression of a superior β-mannanase engineered by cooperative substitution method in Pichia pastoris and its application in preparation of prebiotic mannooligosaccharides. (September 2020)

• Record Type: Journal Article
• Authors: Liu, Zhemin; Ning, Chen; Yuan, Mingxue; Fu, Xiaodan; Yang, Suxiao; Wei, Xinyi; Xiao, Mengshi; Mou, Haijin; Zhu, Changliang
• Is Part Of: Bioresource technology. Volume 311(2020)
• Publication Date: 2020-09
• Subjects: β-mannanase -- Cooperative substitution -- High-efficiency expression -- Prebiotic mannooligosaccharides; Biomass -- Periodicals; Biomass energy -- Periodicals; Bioremediation -- Periodicals; Agricultural wastes -- Periodicals; Factory and trade waste -- Periodicals; Organic wastes -- Periodicals; Bioénergie -- Périodiques; Déchets agricoles -- Périodiques; Déchets industriels -- Périodiques; Déchets organiques -- Périodiques; Déchets (Combustible) -- Périodiques; 662.88
• DOI: 10.1016/j.biortech.2020.123482
• Languages: English
• ISSNs: 0960-8524

Abstract

Graphical abstract: Highlights: A high-specific-activity β-mannanase mutant was gained by cooperative substitution. The expression level of MEIR was superior to most reported β-mannanases. Locust bean gum and konjac gum could be efficiently digested by MEIR. MEIR possesses good potential for prebiotic mannooligosaccharides preparation. Abstract: β-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg −1 ) and MEIR (9003.1 U mg −1 ) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg −1 ), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105, 836 U mL −1 ). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.