title: Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata.
abstract: A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.
authors.affiliation: Department of Food Science, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa 921-8836, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Ishisaki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Honda
authors.forename: Hajime
authors.initials: H
authors.lastname: Taniguchi
authors.forename: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Hamada
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D029748:Caryophyllaceae / Q000201:enzymology*; D020134:Catalytic Domain; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
chemical_list: D009844:Oligosaccharides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002688:Chitinases; D000117:Acetylglucosamine