Enzymatic synthesis of chitooligosaccharides by using chitinolytic Bacillus strains

The enzymatic or chemical depolymerisation of chitin and chitosan delivers chitooligosaccharides (COS). Water soluble, low molecular weight and biologically active COS derivatives are superior to the parent polymers in multiple aspects. This project was designed to produce COS by enzymatic hydrolysis of chitosan using crude chitosanase extracts of previously isolated and identified indigenous chitinolytic Bacillus strains. For this purpose, 16 different chitinolytic Bacillus strains were tested for their chitinase and chitosanase activities. Bacillus mojavensis EGE-B-5.2i (GenBank accession number KC119405) was selected for chitosanase production optimization using statistical methods. A medium containing 7.5 g L⁻¹ soluble starch, 6.5 g L⁻¹ colloidal chitosan, 7.5 g L⁻¹ peptone and 3.5 g L⁻¹ NaCl was discovered and chitosanase activity was enhance up to 9.5 folds using the optimized medium. A chitosanase of 37 kDa was then purified by using ion exchange chromatography. The optimum temperature and pH of purified chitosanase were 55°C and 5.5, respectively. The enzyme was stable at 55°C for 1 h with 66% of its activity and also stable in a wide range of pH (3.0-11.0). The optimum substrate for the chitosanase was soluble chitosan and the Km and Vmax values of enzyme with soluble chitosan were 2.1 mg mL⁻¹ and 244.5 µmol min⁻¹ mg⁻¹, respectively. Furthermore, different columns and detectors were tested to optimize the HPLC method for COS analysis. A gradient elution method was then optimized using DAD detector and Hypersil APS-2 column. The crude chitosanase extracts of Bacillus strains were used to hydrolyse chitosan into COS and the hydrolysates were quantitatively analysed by HPLC using optimized method. Chitosanases of 14 Bacillus strains were able to successfully hydrolyse chitosan to COS and different oligomer from DP 2-6 were detected. Relative yields of the COS products were in a range of 11-31% and chitosanase of Bacillus thuringiensis EGE-B-3.5m has presented maximum relative yield (31%). For most of the chitosanases the major chitosan hydrolysis product was chitobiose. Although the HPLC method was capable to provide efficient separation of COS mixture but for the validation of results and proper characterization of oligomers LC-MS, MALDI-TOF-MS and NMR analysis of the products is necessary.