Purification and Characterization of an Extracellular Chitinase from Bacillus thuringiensis R 176 using Solid State Fermentation with Shrimp Shells Waste

Abstract

The chitinase-producing strain R 176 was isolated from paddy soil in Chiang Mai province, Thailand, and it was identified as Bacillus thuringiensis. The optimal condition of the strain suitable for production of extracellular chitinase was investigated to be a solid state fermentation using a medium (pH 7.0) containing 50 g of shrimp shell powder mixed with 10 mL basal medium (pH 7.0) containing 0.2% (w/v) (NH4)2SO4, 0.1% (w/v) yeast extract, 0.028% (w/v) KH2PO4, 0.025% (w/v) MgSO4.7H2O, and 0.007% (w/v) CaCl2.2H2O. The medium yielded the chitinase of 1.36 U/g IDS, which was 0.36-fold higher than the productivity in a liquid culture with colloidal chitin. The chitinase was purified from the culture broth of strain R 176 by ammonium sulphate precipitation, ion-exchange, and gel filtration. Molecular weight of the chitinase was 40 and 47 kDa compared with standard proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal activity of the purified chitinase was pH 7.0 and 37ºC. More than 80% of R 176 was stable at pH 6.0 to 8.0 and more than 90% at 40ºC. Ca2+ ions, Cu2+ ions and Mg2+ ions inhibited the chitinase activity about 20% and EDTA and p-CMB by 21% and 40%, whereas Ag+ and Zn2+ inhibited the activity up to 65%. Substrate specificity tested indicated that, ball milled chitin (100% relative activity) was the best substrate followed by colloidal chitin (89% relative activity), chitosan, carboxymethyl cellulose, ethylene glycol chitin, glycol chitin and swollen chitin. These results suggested that the substrate specificity of this chitinase was due to the hydrolyzation of glycosidic bond linked between GlcNAc-GlcNAc. For the production of any industrial enzymes, the inexpensive substrates and cost-reducing process like solid state fermentation have been shown several advantages for the upscale production and giving value-added of solid waste.