title: Purification, characterization and physiological significance of a chitinase from the pilei of Coprinopsis cinerea fruiting bodies.
abstract: We purified a chitinase from pilei extractions of Coprinopsis cinerea fruiting bodies by ammonium sulfate precipitation and CM Sepharose cation exchange chromatography. MALDI-TOF/TOF MS analysis characterized this purified chitinase as a putative class V chitinase, ChiB1. ChiB1 hydrolyzed colloidal chitin and chitosan, whereas it did not hydrolyze chitin powder. ChiB1 cleaved only pNP-(GlcNAc)2, rather than pNP-GlcNAc or pNP-(Glc-NAc)3, to release nitrophenol. ChiB1 preferably and progressively released (GlcNAc)2 from (GlcNAc)6 and digested (GlcNAc)6 to two molecules of (GlcNAc)3 in a small proportion, but did not split (GlcNAc)2, so it is an exochitinase. ChiB1 has an optimum temperature range of 35°C to 40°C and an optimum pH of 5.0. ChiB1 exhibited Km and Vmax values of 2.63 mg ml(-1) and 2.31 μmol min(-1) mg protein(-1) for colloidal chitin, respectively. The ChiB1 gene, along with another putative endochitinase (class III chitinase gene), was expressed dominantly among eight predicted chitinase genes in the genome, and its expression level increased with the maturation of fruiting bodies. ChiB1 incubation released a large amount of soluble β-glucan fractions from alkali-insoluble cell wall fractions of C. cinerea fruiting bodies, thereby it may promote the degradation of cell walls in synergy with the β-1,3-glucanases during pileus autolysis.
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: Yajun
authors.initials: Y
authors.lastname: Zhou
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.
authors.forename: Xin
authors.initials: X
authors.lastname: Niu
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: 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 yuansheng@njnu.edu.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yuan
mesh_terms: D000363:Agaricales / Q000737:chemistry / Q000201:enzymology*; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism; D003001:Cloning, Molecular; D048690:Fruiting Bodies, Fungal / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D009596:Nitrophenols; D010940:Plant Proteins; D047071:beta-Glucans; D002686:Chitin; D048271:Chitosan; D002688:Chitinases; C110892:chitinase, class III
keywords: autolysis; cell wall; chitinase; fruiting body; pileus