Masaru Mitsutomi

Family 19 chitinases of Streptomyces species: characterization and distribution.

Chitinase C from Streptomyces griseus HUT6037, described in 1997, is the first family 19 chitinase found in an organism other than higher plants. In this study, some properties of chitinase C were compared with those of family 18 bacterial chitinases, and the distribution of family 19 chitinases in Streptomyces species was investigated. The specific hydrolysing activity of chitinase C against soluble and insoluble chitinous substrates was markedly higher than those of bacterial family 18 chitinases. Chitinase C exhibited marked antifungal activity, whereas the other bacterial chitinases examined had no antifungal activity. Chitinase C was insensitive to allosamidin, whereas the family 18 bacterial chitinases were sensitive. Taking advantage of this insensitivity to allosamidin, a search was made for family 19 chitinases in various Streptomyces species. Chitinases insensitive to allosamidin were detected in the culture supernatants of all tested Streptomyces species. Southern hybridization analysis using a labelled DNA fragment corresponding to the catalytic domain of chitinase C strongly suggested that these species have genes similar to the chiC gene of S. griseus HUT6037. DNA fragments corresponding to the major part of the catalytic domains were amplified by PCR. The amplified fragments encoded amino acid sequences very similar to that of the corresponding region of chitinase C. Therefore, it was concluded that Streptomyces species generally possess family 19 chitinases which are very similar to chitinase C. Comparison of their amino acid sequences with those of plant family 19 chitinases revealed that Streptomyces family 19 chitinases are class IV type in terms of the presence and positions of deletions of amino acid sequences which are characteristic of plant class IV chitinases.

Preparation of chitooligosaccharides from chitosan by a complex enzyme

Chitosan of 24% degree of acetylation was depolymerized by a mixture of cellulase, alpha amylase, and proteinase to give the title oligosaccharides. The removal of products by membrane separation permitted yield maximization of products having degree of polymerization in the 3-10 range.

Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity.

Streptomyces griseus HUT 6037 inducibly produced two chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60°C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanases were obtained for identification of the anomeric form of the reaction products. Both chitosanases produced the β-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 chitosanase with transglycosylation activity.

Functional Analysis of the Chitin-binding Domain of a Family 19 Chitinase from Streptomyces griseus HUT6037: Substrate-binding Affinity and cis-Dominant Increase of Antifungal Function

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBDChiC) differs from those of the plant enzymes. ChBDChiC and the catalytic domain (CatDChiC), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBDChiC binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBDChiC resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBDChiC itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatDChiC in trans.

Immobilization of thermostable α-galactosidase from Pycnoporus cinnabarinus on chitosan beads and its application to the hydrolysis of raffinose in beet sugar molasses

Abstract α-Galactosidase (EC 3.2.1.22) from Pycnoporus cinnabarinus was immobilized on chitosan beads, BCW 1000, and crosslinked chitosan beads, BCW 3000 and 3500, of three different sizes, which were untreated or previously treated with glutaraldehyde. The activity yields of the immobilized enzymes were between 25 to 45%, except for glutaraldehyde-untreated B BCW 1000. Leakage of the enzyme with increasing ionic strength was observed in glutaraldehyde-untreated BCW 1000 and 3000. The α-galactosidases immobilized on glutaraldehyde-treated BCW 3000 and 3500 were active at pH 3–6 and at 70–80°C, and stable between pH 3 and 9, and below 70°C. The immobilized α-galactosidase was continuously used for 30 days to hydrolyze raffinose in beet sugar molases.

Two exo-β-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases

A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.

Family 19 Chitinases from Streptomyces thermoviolaceus OPC-520: Molecular Cloning and Characterization

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70°C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.

Purification and characterization of a 49-kDa chitinase from Streptomyces griseus HUT 6037

A 49-kDa chitinase (pI7.3) was purified to homogeneity from the culture supernatant of Streptomyces griseus HUT 6037 by ultrafiltration, DEAE-Sephadex A-50 and Sephadex G-100 column chromatographies, and chromatofocusing. The purified enzyme was stable up to 40 degrees C. The N-terminal amino acid sequence of the enzyme was highly homologous to the N-terminal region of the fibronectin type III-like domain of S. olivaceoviridis chitinase 01 belonging to family 18 glycosyl hydrolases. The 49-kDa chitinase hydrolyzed partially N-acetylated chitosan more easily than colloidal chitin. The hydrolyzate of 54% deacetylated chitosan by the enzyme was separated by CM-Sephadex C-25 column chromatography. The structures of the oligosaccharides obtained were determined by MALDI-TOF MS analysis combined with exo-glycosidase digestion. In addition to GlcNAc, (GlcNAc)2, and (GlcNAc)3, hetero-chitooligosaccharides with GlcNAc at the reducing end were detected. Thus, the specificity of the enzyme for the hydrolysis of the beta-1,4-glycosidic linkages in partially N-acetylated chitosan was similar to that of the family 18 chitinases.

Synthesis of a chitosan tetramer derivative, β-d-GlcNAc-(1→4)-β-d-GlcNAc-(1→4)-β-d-GlcNAc-(1→4)-d-GlcN through a partial N-acetylation reaction by chitin deacetylase

Abstract We have synthesized β- d -GlcNAc-(1→4)-β- d -GlcNAc-(1→4)-β- d -GlcNAc-(1→4)- d -GlcN (2) through a partial N-acetylation reaction of chitosan tetramer 1 by a chitin deacetylase from Colletotrichum lindemuthianum ATCC 56676. The compound was purified from the mixture of acetylation products of 1 using cation-exchange column chromatography and amine-adsorption column chromatography, and its structure was estimated by 1H NMR and FABMS analyses. The enzymatic reaction allows a regioselectivity that is hard to achieve by chemical N-acetylation.

Purification and characterization of novel chitinases from Streptomyces griseus HUT 6037

Abstract Two chitinases, C-1 and C-2, were purified from the culture supernatant of Streptomyces griseus HUT 6037 by ammonium sulfate precipitation, Butyl-Toyopearl 650M chromatography, and chromatofocusing. Both enzymes had a molecular weight estimated to be 27,000 by SDS polyacrylamide gel electrophoresis, while their p I s were 7.7 and 7.3, respectively. The enzymes were active from pH 4.5 to 6.0 and their optimum temperature was 55°C. They were stable between pH 6.5 and 10.0 and at temperatures below 50°C. Chitinases C-1 and C-2 hydrolyzed chitin, colloidal chitin, glycol chitin, carboxymethyl chitin, 53% deacetylated chitosan, and (GlcNAc) 3–6 , but did not hydrolyze 96% deacetylated chitosan and (GlcN) n . The hydrolyzates of 53% deacetylated chitosan by these enzymes were separated by CM-Sephadex C-25 column chromatography. The products were analyzed structurally to elucidate the specificity of the chitinases. The oligosaccharides isolated had GlcNAc as the nonreducing end and GlcNAc or GlcN as the reducing end residues. These results indicate that the enzymes cleave both the N -acetyl-β- d -glucosaminidic and the β-glucosaminidic linkages in partially N -acetylated chitosan molecules.