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

Zhemin Liu; Chen Ning; Mingxue Yuan; Xiaodan Fu; Suxiao Yang; Xinyi Wei; Mengshi Xiao; Haijin Mou; Changliang Zhu

doi:10.1016/j.biortech.2020.123482

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

Bioresour Technol. 2020 Sep:311:123482. doi: 10.1016/j.biortech.2020.123482. Epub 2020 May 5.

Authors

Zhemin Liu¹, Chen Ning¹, Mingxue Yuan¹, Xiaodan Fu¹, Suxiao Yang¹, Xinyi Wei¹, Mengshi Xiao¹, Haijin Mou², Changliang Zhu³

Affiliations

¹ College of Food Science and Engineering, Ocean University of China, Qingdao 266003 China.
² College of Food Science and Engineering, Ocean University of China, Qingdao 266003 China. Electronic address: mousun@ouc.edu.cn.
³ College of Food Science and Engineering, Ocean University of China, Qingdao 266003 China. Electronic address: chlzhu@163.com.

PMID: 32416491
DOI: 10.1016/j.biortech.2020.123482

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.

Keywords: Cooperative substitution; High-efficiency expression; Prebiotic mannooligosaccharides; β-mannanase.

MeSH terms

Amorphophallus*
Hydrolysis
Pichia
Prebiotics
beta-Mannosidase*

Substances

Prebiotics
beta-Mannosidase