Biochemical and Genetic Properties of PaenibacillusGlycosyl Hydrolase Having Chitosanase Activity and Discoidin Domain

Abstract

Cells of "Paenibacillus fukuinensis" D2 produced chitosanase into surrounding medium, in the presence of colloidal chitosan or glucosamine. The gene of this enzyme was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids and its molecular weight was 85,610. Unlike conventional family 46 chitosanases, the enzyme has family 8 glycosyl hydrolase catalytic domain, at the amino-terminal side, and discoidin domain at the carboxyl-terminal region. Expression of the cloned gene in Escherichia coli revealed ␤-1,4-glucanase function, besides chitosanase activity. Analyses by zymography and immunoblotting suggested that the active enzyme was, after removal of signal peptide, produced from inactive 81-kDa form by proteolysis at the carboxyl-terminal region. Replacements of Glu 115 and Asp 176 , highly conserved residues in the family 8 glycosylase region, with Gln and Asn caused simultaneous loss of chitosanase and glucanase activities, suggesting that these residues formed part of the catalytic site. Truncation experiments demonstrated indispensability of an amino-terminal region spanning 425 residues adjacent to the signal peptide.

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

“…The deduced amino acid sequences of the cloned celC have been shown to involve similar GH-8 conserved residues with the following pattern: (Figure 2b). Glutamic acid and aspartic acid have been recognized as conserved catalytically active amino acid residues in GH-8 [77,78] glutamic acid active site residue was suggested to act as a proton donor in the catalytic reaction, while the aspartic acid residue was inferred to have a catalytic role as a nucleophile [79]. The significant ablation that has been observed in the enzymatic activity upon the replacement of either active-site residue by site-directed mutagenesis emphasizes their main catalytic role [77].…”

Uncoupling chitosanase production from chitosan

“…Induction by chitosan was specific, as P. fukuinensis D2 failed to produce chitosanase in the presence of CM-cellulose or lichenan. 9 In a few cases, the native strains produced chitosanase in the absence of chitosan. The GH8 chitosanase from Bacillus sp.…”

“…Examples of well studied chitosanases purified directly from the culture supernatants of the native strains include the GH46 chitosanase from Bacillus circulans MH-K1 8 and the GH8 chitosanase from Paenibacillus fukuinensis D2. 9 Addition of chitosan to the culture medium was required in both cases. The production of chitosanase by P. fukuinensis D2 was strongly repressed in the presence of glucose.…”

Activity Measurement of Chitosanase by an Amperometric Biosensor

“…1 In this work, the catalytic domain of the chitosanase, deficient in the discoidin domains (COOH-terminal 271-amino acid region), 2 was used. The specific activity of the enzyme was determined to be 20.1 units mg -1 by the conventional method described above with glycol chitosan as a substrate.…”

“…Recently, one of the authors (H. Kimoto) newly found a chitosanase that belongs to the family 8 glycosyl hydrolase group, in a Paenibacillus fukuinensis D2 culture supernatant. 1 This enzyme is expected to be a tool for manufacturing chitosan oligosaccharides with potential antibacterial, anticancer, or elicitor activity. 2 So far, the activity of the chitosanase has been assayed by the detection of amino sugars (here, glucosamine and chitosan oligosaccharides) released in the reaction mixture of chitosan and chitosanase after incubation for 10 -120 min at 37°C.…”