Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus

• Published in: The journal of microbiology Vol. 51; no. 2; pp. 189 - 193
• Main Authors: Kang, Yuri, Kim, Hyewon, Choi, Hyoung T
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer-Verlag 01.04.2013; The Microbiological Society of Korea; Springer Nature B.V; 한국미생물학회
• Subjects: active sites; Agaricales - chemistry; Agaricales - enzymology; Agaricales - genetics; Agaricales - growth & development; Amino Acid Sequence; amino acid sequences; Amino acids; Antifungal agents; autolysis; Biomedical and Life Sciences; cell walls; chitin; Chitinase; Chitinases - chemistry; Chitinases - genetics; Chitinases - isolation & purification; Chitinases - metabolism; Cloning; Cloning, Molecular; complementary DNA; Coprinellus; droplets; Enzyme Stability; Enzymes; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; Fungi; glucans; Hydrogen-Ion Concentration; Kinetics; Life Sciences; Microbial Physiology and Biochemistry; Microbiology; Microscopy; Molecular Sequence Data; molecular weight; Mushrooms; nucleotides; Pichia pastoris; Polymerase chain reaction; rapid amplification of cDNA ends; Substrate Specificity; temperature; 생물학; United States > US; South Korea; mushroom autolysis; chitinase
• ISSN: 1225-8873; 1976-3794
• DOI: 10.1007/s12275-013-2535-9

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⁻¹unit⁻¹, respectively.