Screening of active sites and study on immobilization of Bacillus cereus phospholipase C

Abstract

Bacillus cereus phospholipase C (PLCBC) was obtained by treating Bacillus cereus with ultraviolet light and diethyl sulfate. The active centers of PLCBC were determined, and its inactive center groups were combined with the carboxyl modified carrier to obtain immobilized PLCBC. The enzyme activity was 289 U/mg. The active group of the enzyme was –COOH, while the binding site was –NH2. The –COOH and –NH2 of PLCBC were chemically modified. The immobilization sites –COOH and –NH2 were used to covalently bind to the carrier, and the enzyme activity recovery ratio was 59.0% and 95.0%, respectively, with Vmax and Michaelis constant of the latter being closer to the free PLCBC. The carrier and immobilized enzyme were characterized. The optimal pHs for the free and immobilized PLCBC were 7.0 and 7.5, respectively, and the optimal temperatures were 50 °C and 55 °C, respectively. After 7 cycles, the relative activity of PLCBC–Fe3O4/SiO2/HPG–COOH was 87%. In this work, a new type of immobilized PLCBC was prepared through active site screening and oriented immobilization. It provided a theoretical basis for the application of immobilized phospholipase in industrial oil degumming.