title: Rational design for the stability improvement of Armillariella tabescens β-mannanase MAN47 based on N-glycosylation modification.
abstract: β-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant β-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation. The mutant enzymes were expressed in Pichia pastoris SMD1168, and their thermal stability, pH stability, trypsin resistance and pepsin resistance were determined. Two mutant MAN47 enzymes, g-123 and g-347, were glycosylated as expected when expressed in yeast, and their thermal stability, pH stability, and protease resistance were significantly improved compared to the wild-type enzyme. An enzyme with multiple stability characterizations has broad prospects in practical applications, and the rational design N-glycosylation strategy may have applications in simultaneously improving several properties of other biotechnological targets.
authors.affiliation: Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China.
authors.forename: Weixiong
authors.initials: W
authors.lastname: Hu
authors.affiliation: Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Liu
authors.affiliation: Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Li
authors.affiliation: Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China; Department of Bioengineering, Jinan University, Guangzhou City, Guangdong Province 510632, China.
authors.forename: Daling
authors.initials: D
authors.lastname: Liu
authors.affiliation: National Engineering Research Center of Genetic Medicine, Guangzhou City, Guangdong Province 510632, China.
authors.forename: Zhihe
authors.initials: Z
authors.lastname: Kuang
authors.affiliation: National Engineering Research Center of Genetic Medicine, Guangzhou City, Guangdong Province 510632, China.
authors.forename: Chuiwen
authors.initials: C
authors.lastname: Qian
authors.affiliation: Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province 510632, China; National Engineering Research Center of Genetic Medicine, Guangzhou City, Guangdong Province 510632, China. Electronic address: tdsyao@jnu.edu.cn.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Yao
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology; D020134:Catalytic Domain; D000068016:Eubacteriales / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006031:Glycosylation; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: Enzyme stability; Homology modeling; Molecular dynamics simulations; N-glycosylation; Posttranslational modification