Yong Chul Shin

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.

Effect of Sparassis crispa Extracts on Immune Cell Activation and Tumor Growth Inhibition

Sparassis crispa is an edible mushroom with medicinal properties that contains more than 40% βglucan. The role of S. crispa in regulating the functional activation of macrophages has yet to be fully elucidated. The objective of this study was to investigate the molecular mechanism underlying the immune-stimulatory function of S. crispa soluble β-glucan and extracts on macrophages. In this study, we showed that S. crispa soluble β-glucan was able to stimulate TNF-α and IL-1β production through NF-kB activation in Raw 264.7 cells. We also showed that S. crispa extracts could not only enhance TNF-α production in Raw 264.7 cells, but also suppress tumor growth in vivo. All of our results suggest that S. crispa could be developed as a promising immunostimulatory principle, applicable to cancer patients.Sparassis crispa is an edible mushroom with medicinal properties that contains more than 40% β-glucan. The role of S. crispa in regulating the functional activation of macrophages has yet to be fully elucidated. The objective of this study was to investigate the molecular mechanism underlying the immune-stimulatory function of S. crispa soluble β-glucan and extracts on macrophages. In this study, we showed that S. crispa soluble β-glucan was able to stimulate TNF-α and IL-1β production through NF-kB activation in Raw 264.7 cells. We also showed that S. crispa extracts could not only enhance TNF-α production in Raw 264.7 cells, but also suppress tumor growth in vivo. All of our results suggest that S. crispa could be developed as a promising immunostimulatory principle, applicable to cancer patients.