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A chitosanase gene, designated to choA encoding one of chitosanases of Matsuebacter chitosanotabidus 3001 was cloned in Escherichia coli. In order to construct the chitosanase gene into an high-level expression vector, the gene choA was subcloned into an ITPG inducible vector pFLAQ. The recombinant plasmid DNA pFLAQ: choA was introduced into E. coli DH5a. Chitosanase (34-kDa) was successfully overexpressed in E. coli cell harboring the recombinant pFLAQ: choA. A highly sensitive and selective high-performance liguid chromatography (HPLC) has been developed for the analysis of chitooligosaccharides. The hydrolyzates were derivatized with PMP (1-phenyl-3-nethyl-5-pyrazolone), and its were quantitated by HPLC with monitoring in UV absorbance at 245 nm. Chitosan oligosaccharides, (GlcN)n, n=2~4 and some soluble materials (>5), were produced by using highly expressed chitosanase involved in crude extract which was partially Purified. GlcN2-4 was mainly observed in the reaction mixture, whereas purified chitosanase mainly producing GlcN2. Furthermore, GlcN2-4 was purified in gam-scale using an anion exchange column Dowex50WX8-200 eluted by gradient from 0 to 4.0 M ammonium formate(PH 4.5). In addition, purified chitosan oligosaccharides (GlcN)n, n=2-4 were acetylated with 3H-acetic anhydride for further study.
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