Gene Cloning,Expression And Activity Analysis Of Chitosanase From Bacillus Amyloliquefaciens HZ-1510

Chitosan can be produced by chitin which is the second most abundant polymers in the nature only after the cellulose. The hydrolysis products from chitosan have been widely used in medicine,agriculture and industry. For example, oligochitosan (its hydrolysis product) can enhance the immune responses of fish and is a promising addition of fish feed. However, the high molecular weight and low solubility of chitosan have hindered its wide utilization. Therefore, the knowledge about the efficient hydrolysis of chitosan can not only improve the application of chitosan, but also lay the theoretical foundation for the manufacture of oligochitosans. Bacillus amyloliquefaciens is a member of the genus Bacillus, which exists widely in nature. We have screened and obtained a bacterial strain which could grow on a selective medium with chitosan as the sole carbon source. It was identified as Bacillus amyloliquefaciens. In this study, we focused our studies on the activity of chitosanase of B. Amyloliquefaciens. The major results are follows:1.Basing on the physiological and biochemical features, as well as 16S rDNA and gyrA gene sequences. The strain was identified as Bacillus amyloliquefaciens HZ-1510.2.The chitosanase gene of Bacillus amyloliquefaciens HZ-1510 was cloned and analyzed.It consisted of an open reading frame of 837 bp which encoded a protein of 279 amino acids with predicted molecular weight of 31.45 kDa. The protein contained a signal peptide with a cleavage site located between the 36th and 37th amino acids. The deduced amino acid sequence homology analysis of the chitosanase revealed that it belonged to GH46 family, and had the highest similarity with those from B. amyloliquefaciens strain ABBD and B. amyloliquefaciens strain MBE1283. Furthermore, two amino acids (Glu-55 and Asp-71) are the catalytic active sites.3.The glutathione S-transferase (GST) - chitosanase fusion protein expression plasmid pGEX-5x-1-Chi was constructed, and it was expressed in Escherichia coli BL21. The recombinant chitosanase has been purified by using GST column chromatography.4.The optimal pH and temperature for the chitosan hydrolysis activity was 5.5 and 55 ?,respectively. The enzyme activity was increased in the presence of Mn2+,Ca2+ and Mg2+.However, Fe3+, Ag+, Cu2+, Ba2+ and K+ could inhibit its activity. Furthermore, its activity could be slightly increased in the presence of 0.1 mmol/L Zn2+ while it was inhibited by 2.0 mmol/L Zn2+.