Molecular Characterization of a Novel Family-46 Chitosanase fromPseudomonassp. A-01

Abstract

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.

Classification of Chitosanases by Hydrolytic Specificity towardN1,N4-Diacetylchitohexaose

“…CsO-2 33) and Pseudomonas sp. A-01 8) have been isolated from soil. These strains possess strong chitosan-degrading activity.…”

“…CsO-2 4,34) and Pseudomonas sp. A-01, 8) but the hydrolytic specificity of neither has been studied. Hence, we attempted to clarify the hydrolytic specificities of both enzymes by our new method using chitohexaose and N 1 ,N 4 -diacetylchitohexaose as substrates.…”

“…Chitosanases (EC 3.2.1.132) are glycosyl hydrolases that catalyze the endohydrolysis of -1,4 linkages between GlcN residues in a partially Nacetylated chitosan (Enzyme Nomenclature, http:// www.chem.qmul.ac.uk/iubmb/enzyme/). They are produced by various bacteria [1][2][3][4][5][6][7][8][9] and fungi, [10][11][12][13][14][15] and are useful in the preparation of biofunctional chitooligosaccharides. Fukamizo et al 16) have proposed that chitosanases can be classified into three subclasses according to their specificity for the hydrolysis of the -glycosidic linkages in partially N-acetylated chitosan molecules: subclass I chitosanases [16][17][18][19][20] split both GlcN-GlcN and GlcNAc-GlcN bonds, subclass II chitosanases 3) split only the GlcN-GlcN bond, and subclass III chitosanases 5,[21][22][23] split both GlcN-GlcN and GlcN-GlcNAc bonds.…”

Chitosan degradation and associated changes in bacterial community structures in two contrasting soils

“…A-01, Amycolatopsis sp. CsO-2 and Bacillus circulans MH-K1; Ando et al 2008;Saito et al 2009;Tomita et al 2013).…”

High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris

“…According to the carbohydrate-active enzymes database (www.cazy.org), chitosanases are classified into five glycoside hydrolase families: GH-5, GH-8, GH-46, GH-75 and GH-80. Among these, GH-5 and GH-8 act on glycosides as well as chitosan, whereas GH-46, GH-75 and GH-80 are chitosanases attacking chitosan (Ando et al 2008). Xiaomei Chen and Chao Zhai contributed equally to this work.…”