Purification and Characterization of Chitosanase from Bacillus sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides

Abstract

For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.Chitosan is currently obtained by the deacetylation of chitin (poly-␤-1,4-D-N-acetylglucosamine) that has been extracted from an abundant source of shrimp or crab shells. Deacetylated chitosans are produced by treating chitin in a concentrated alkaline solution (50%, wt/vol) and boiling it for several hours (34). Chitosan is also found in nature; it is found in the cell walls of fungi of the class Zygomycetes, in the chlorophycean algae Chlorella sp. (26), and in insect cuticles (2). These natural chitosans are synthesized by the tandem action of chitin synthetase and chitin deacetylase, as shown for Mucor rouxii and Colletotrichum lindemuthianum (9). Both chemical and enzymatic procedures for chitosan production result in the incomplete deacetylation of chitin, which yields chitosans in the intermediate range of the degree of deacetylation (DDA). Consequently, chitosan can be considered as a partly deacetylated derivative of chitin; it must have diverse structures containing hetero-linkages of N-acetylglucosamine (GlcNAc)-glucosamine (GlcN) and Gl...

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].…”

Chitosanase production by Paenibacillus ehimensis and its application for chitosan hydrolysis

“…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.…”

Production and Characterization of Partially Purified Chitosanase Enzyme of <i>Bacillus cereus</i> A4/B4 Isolated from Biowaste Soil Samples:Bioactive Chitooligosaccharide

“…The chitosanase activity was higher in glucose [22] and starch [10] containing medium. The bacterial colonies utilized 1% glucose, 1% fructose and 1% xylose as a major carbon source and showed higher enzyme activity (Figure 4).…”

“…From the suspension 0.1ml was spread plated on to the CDA [6] plates having basal medium M9 along with (0.5% ,1% ,2%) substrate chitosan or colloidal chitosan of 0.5% and 1% [7][8][9], Minimal Salt Chitosan (MS) medium [10,11], Minimal Synthetic (MSM) Medium [12] and all the plates were incubated at 30° C for 3-5 days.…”