Purification, Characterization, and Gene Cloning of a Chitosanase from Bacillus Species Strain S65

Abstract

For the production of oligosaccharides from chitosan, a chitosanase-producing bacterium, S65, was isolated from soil. On the basis of phylogenetic analysis of the 16S rDNA gene sequence and phenotypic analysis, S65 was identified as a Bacillus sp. strain. This bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. S65 chitosanase was homogeneously purified by DEAE Sepharose fast flow anion exchange followed by Superdex 75 size exclusion, and the molecular weight was 45 kDa according to SDS−PAGE. Enzyme analysis showed that the optimum pH and temperature of S65 were 6.0 and 65 °C, respectively. Catalytic activity was stable from pH 5.5−6.5 at temperatures below 40 °C, and the pI of chitosanase was about 6.0 as determined by a test tube method. S65 chitosanase degraded carboxymethyl cellulose (CMC) at the degree of about 5.3% relative to the value of soluble chitosan, but it cannot hydrolyze colloidal chitin and crystalline cellulose. Gene encoding was cloned and sequenced. The deduced amino acid sequence of the S65 exhibited the highest homology to those of family 8 glycanase, suggesting that the enzyme belonged to family 8. Keywords: Chitosan; chitosanase; purification; characterization; gene cloning

Chitosanase production by Paenibacillus ehimensis and its application for chitosan hydrolysis

“…strains showed optimum activities at pH values between 4.0 and 8.0, the optimum temperatures of activity have been reported to be in the ranges 30-70°C (Kurakake et al 2000;Aktuganov et al 2003;Jo et al 2003;Choi et al 2004;Kim et al 2004;Su et al 2006;Gao et al 2008;Wang and Yeh, 2008). The effect of pH and temperature on the catalytic activity of chitosanase from P. ehimensis was studied by using soluble chitosan as substrate changing the pH and temperature during the assay.…”

“…Enzymatic hydrolysis has some advantages in producing COS. Chitosanases can catalyze the hydrolysis under mild conditions (Kuo et al 2004;Ming et al 2006) Chitosanases have been recognized as enzymes that attack chitosan but not chitin and defined as the enzyme performing endohydrolysis of β-1,4-linkages between D-glucosamine residues in a partly acetylated chitosan (Su et al 2006). Chitosanase has been found in a variety of microorganisms, including various bacterial species (Kurakake et al 2000;Choi et al 2004;Kim et al 2004;Su et al 2006;Sun et al 2007;Zhu et al 2007;Gao et al 2008;Wang and Yeh, 2008;Wang et al 2008a;Wang et al 2008b) and fungi Ike et al 2007). Although, microbial chitosanases shows excellent performances in COS production, they are expensive to be utilized in large-scale industrial applications, because enzyme hydrolysis requires multi-steps, particularly, enzyme preparation and purification.…”

Optimization of chitosanase production from Bacillus sp. RKY3 using statistical experimental designs

“…RKY3 was high compared with Bacillus sp. S65 (1.45 U mL −1 ) 13 , Acinetobacter sp. C-17 (2.80 U mL −1 ) 30 , Gongronella sp.…”

“…Thus, the production of chitooligosaccharides from chitosan has received increased attention in the nutraceutical and medical industries. 1,13 Chitooligosaccharides can be produced both by chemical and enzymatic methods. The enzymatic hydrolysis methods, however, have recently become more and more popular, as chemical methods such as acid hydrolysis typically result in the production of a large quantity of short-chain oligosaccharides and low yields of oligosaccharides, which necessitates additional unit steps for the isolation of high molecular weight oligosaccharides.…”

“…The enzymatic hydrolysis methods, however, have recently become more and more popular, as chemical methods such as acid hydrolysis typically result in the production of a large quantity of short-chain oligosaccharides and low yields of oligosaccharides, which necessitates additional unit steps for the isolation of high molecular weight oligosaccharides. 13,14 The enzymatic hydrolysis of chitosan for the production of chitooligosaccharides is generally conducted using chitosanase (EC 3.2.1.132), which catalyzes the endohydrolysis of β-1,4-linkages between D-glucosamine residues in a partially deacetylated chitosan, including GlcN-GlcN, GlcN-GlcNAc, and GlcNAc-GlcN. 15,16 Chitosanases have previously been produced by a number of organisms, including fungi, 17 plants, 18 and bacteria, 16,19,20 but the bacterial chitosanases detected in Bacillus species are currently the most frequently investigated.…”