The Research Of Chitinase From Streptomyces Sp. DA11 Associated With South China Sea Sponge Craniella Australiensis

In this paper, statistical Plackett–Burman design and Box-Bohnken Response Surface Methodology were applied to optimize the medium components to improve the chitinase activity of Streptomyces sp. DA11 associated with South China Sea sponge Craniella australiensis. Firstly, galactose and peptone were found to be the suitable carbon and nitrogen sources for the growth and chitinase activity by single factor Seriatim-Factorial test. Secondly, galactose, colloid chitin, MgSO4·7H2O were proved to have remarkable effects on chitinase activity. Finally, an optimal medium was obtained by Box-Bohnken methodology, which consisted of 5.00 g/L galactose, 2.62 g/L colloid chitin, 0.10 g/L MgSO4·7H2O and 12.5 g/L peptone, 1.5 g/L PO43- (KH2PO4 0.45 g/L, K2HPO4 1.05 g/L), 12.5 g/L powder chitin, 0.03 g/L FeSO4 and 0.03 g/L ZnSO4·7H2O with artificial sea water (ASW). With this optimal medium, both the chitinase activity and cell growth were remarkably enhanced. The chitinase activity of 1559.2 U/g cell dry weight (36.43U/mL) and the maximum cell dry weight of 23.3 g/L were reached after 72 h, which was 39.2-fold and 2.6-fold higher than that of the basic medium, respectively. Through purification by 80% ammonium sulfate followed by affinity binding to chitin and DEAE-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg-1 was achieved and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of ca. 34 kDa. For the purified chitinase, antifungal activity was observed against Aspergillus niger and Candida albicans, which indicated the antifungal potential for biomedicine. The pH, temperature and salinity optima of the enzyme were 8.0, 50℃and 45 g‰psu, respectively. Thermal stability, pH and salinity tolerance of the chitinase from Streptomyces sp. DA11 may contribute to its potential application compared with land chitinase. Meanwhile, chitinase activity can be increased by metal ions Mn2+, Co2+, Cu2+, Zn2+ and Mg2+, while strongly inhibited by Ca2+, Fe2+ and Ba2+. With colloidal chitin as substrate instead of powder chitin, higher Vmax (0.82 mg product/min·mg protein) and lower Km (0.019 mg/mL) values can be achieved suggesting that colloidal chitin was a better substrate for the enzyme.