1. High-efficiency expression of a superior β-mannanase engineered by cooperative substitution method in Pichia pastoris and its application in preparation of prebiotic mannooligosaccharides
- Author
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Chen Ning, Mingxue Yuan, Suxiao Yang, Mengshi Xiao, Zhemin Liu, Xiaodan Fu, Xinyi Wei, Changliang Zhu, and Haijin Mou
- Subjects
0106 biological sciences ,Environmental Engineering ,medicine.medical_treatment ,Mutant ,Bioengineering ,Industrial fermentation ,010501 environmental sciences ,01 natural sciences ,Pichia ,Pichia pastoris ,010608 biotechnology ,medicine ,Recombinase ,Waste Management and Disposal ,0105 earth and related environmental sciences ,biology ,Renewable Energy, Sustainability and the Environment ,Chemistry ,Hydrolysis ,Prebiotic ,beta-Mannosidase ,General Medicine ,biology.organism_classification ,Enzyme assay ,Prebiotics ,Biochemistry ,biology.protein ,Fermentation ,Heterologous expression ,Amorphophallus - 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 (
- Published
- 2020