title: Gene cloning, heterologous expression and characterization of a Coprinopsis cinerea endo-β-1,3(4)-glucanase.
abstract: A gene coding endo-β-1,3(4)-glucanase (ENG16A) was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris. ENG16A only acts on substrates containing β-1,3 glycosidic bonds but not on substrates containing only β-1,4- or β-1,6-glycosidic bonds. Interestingly, compared to the activity of this enzyme towards carboxymethyl (CM)-pachyman containing only β-1,3-glycosidic bonds, its activity towards barley β-glucan containing both β-1,3-glycosidic and β-1,4-glycosidic bonds was increased by 64.72 %,, its activity towards laminarin containing both β-1,3-glycosidic and β-1,6-glycosidic bonds was decreased by 50.83 %. In addition, ENG16A has a higher Km value and Vmax for barley β-glucan than laminarin, which may be related to the fact that barley β-glucan contains mainly β-1,4-glycosidic bonds mixed with a few β-1,3-glycosidic bonds, whereas laminarin mainly contains β-1,3-glycosidic bonds with a few β-1,6-branched glucose residues. The adjacent β-1,4-glycosidic bond promotes ENG16A to hydrolyse β-1,3-glycosidic bonds, leading to an increased Vmax; the nearby β-1,6-glycosidic bonds inhibited its hydrolysis of β-1,3-glycosidic bonds, resulting in a decreased Vmax. This property is suggested to be related to the mechanism that C. cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.
authors.affiliation: Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China.
authors.forename: Liqin
authors.initials: L
authors.lastname: Kang
authors.affiliation: Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China. Electronic address: liuzhonghua8501@163.com.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China. Electronic address: yuansheng@njnu.edu.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yuan
mesh_terms: D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D044644:Endo-1,3(4)-beta-Glucanase
keywords: Barley β-glucan; Glucanase; Laminarin; Nutrition degradation; Wall autolysis