title: Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus.
abstract: Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was used as the substrate. The K m value and V max for the substrate A, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min(-1)unit(-1), respectively.
authors.affiliation: Department of Biochemistry, Kangwon National University, Chunchon 200-701, Republic of Korea.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kang
authors.forename: Hyewon
authors.initials: H
authors.lastname: Kim
authors.forename: Hyoung T
authors.initials: HT
authors.lastname: Choi
mesh_terms: D000363:Agaricales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D002688:Chitinases