Immobilization of Haloalkalophilic Lipase from Bacillus Cereus Ms6 Bacteria and its Characterization

Lipases belong to the class of hydrolases which are able to catalyze the hydrolysis of long chain triglycerides and many other reactions including ester synthesis and transesterification in organic media containing minute concentration of water. The immobilization of enzyme is an important strategy to improve desirable features of enzymes besides enhancing the stability of the enzyme and to obtain additional beneficial properties via immobilization process. In addition, the immobilized enzyme could be used repeatedly or continuously. In our study, the isolated lipase produced by Bacillus cereus MS6 was immobilized on 3% sodiumalginate beads by the entrapment methods. The alginate bead was prepared as an aqueous mixture of sodium alginate, the enzyme and CaCl2 to increase its reusability, and overall enzyme stability. Various parameters such as alginate and CaCl2 concentration, lipase concentration loading and bead size were evaluated for the optimum immobilization yield. It was observed that with an increase in alginate concentration, the yield of immobilized enzyme was also increased up to a certain limit. A similar case was observed with CaCl2 addition, the optimum concentrations of alginate and CaCl2 were observed to be 3% and 2% (w/v), respectively. The maximum production and activity of immobilized lipase were 180 mg/ml and 550 U/ml, respectively with an observed yield of 88.70 %, while the maximum activity of free enzyme was 450 U/ml with an activity yield of 40%. An increased beads size resulted in the decrease of the yield of immobilization. These lipase immobilized beads could be reused for many cycles for the hydrolysis of triglycerides without any loss in the activity. The entrapped lipase was more stable over a wide range of temperatures, pH, and storage time as compared to free enzyme. The catalytic properties of the immobilized lipase were also compared with that of the free enzyme. The optimum pH of the free enzyme was found to be 9.0 at a temperature maximum of 400C, while that the immobilized of lipase was pH10.0 at a temperature maximum of 500C. The optimum substrate concentration for free and immobilized enzyme was found to be 2% and 3%, respectively. The overall characterization data reveals an increase in the stability of the immobilized lipase as compared to free lipase.