Sulfolobus solfataricus P2 β-半乳糖苷酶在枯草芽孢杆菌中的表达及固定化

Expression of Sulfolobus solfataricus P2 β-galactosidase in Bacillus subtilis and study of immobilization

研究室获得1株来源于Sulfolobus solfataricus P2的β-半乳糖苷酶突变体F441Y,该突变体具有转苷性高,热稳定性好的特点。研究中,成功将β-半乳糖苷酶突变体F441Y在枯草芽孢杆菌中进行重组表达,3 L发酵罐发酵80 h后胞内酶活达到76.5 U/m L。鉴于β-半乳糖苷酶为胞内表达,为提高酶的使用效率并降低酶的成本,以2%的海藻酸钠、1%的明胶混合作为固定化包埋载体,2%氯化钙为固化液,0.5%的戊二醛为交联剂对重组菌进行细胞的固定化,全细胞酶活回收率达到62%,将该固定化细胞进行乳糖的酶转化,优化酶转化条件后,低聚半乳糖的最高转化率高达55.1%。

A Sulfolobus solfataricus P2 β-galactosidase mutant F441 Y was constructed,which has the characteristics of high trans-galactosyl activity and good thermostability. In this study,β-galactosidase mutant F441 Y was successfully expressed in Bacillus subtilis and the fermentation process was optimized. After 80 h fermentation in 3 L fermentor,the intracellular enzyme activity reached 76. 5 U/m L. Since β-galactosidase is expressed intracellularly,it was immobilized to increase the enzyme usage efficiency and reduce the cost. The immobilization of recombinant β-galactosidase cells was performed using 2% sodium alginate and 1% gelatin as carrier,2% calcium chloride as curing solution,and 0. 5% glutaraldehyde as cross-linking agent. The enzyme activity recovery rate of the cell immobilization reached 62%. Using the immobilized cells for lactose enzyme conversion,the highest conversion of galactooligosaccharides was up to 55. 1% after optimizing the conversion conditions.