Expression And Secretion Of Thermostable Lactase

β-Galactosidase (EC.3.2.1.23), also named lactase, which can hydrolyzeβ-1,4-D-galactosidic linkage, is industrially important for applications in producing lactose-free milk which can eliminate the lactose-intolerance of human beings and accelerate the rapid development of dairy industry. Compared to the mesophilic enzyme, thermostableβ-galactosidases have great superiority in saving energy and equipment, making operations within one shift.The thermostableβ-galactosidase encoded by gene bgaB from Bacillus stearothermophilus has good thermostability ,and the enzyme acitivity is stable even at 70℃.The commerical enzymes with enormous industrial application, such as thermostableβ-galactosidase, were commonly produced by secretion of the enzyme into fermentation supernatant, which could greatly simplify the progress of extraction, isolation and purification and reduce the manufacturing costs. However, our laboratory has failed to secrete thermostableβ-galactosidase by some signal peptides, including penicillin acylase, amylase, neutral protease in B. subtilis. It was probably that the special structure in BgaB prevented its secretion. Hence, the object of this paper was to investigate the influence of the structure in BgaB on its secretion, and to attempt to improve the secreting efficiency of BgaB by altering the stair structure.The protein expression is before secretion, therefore this paper tried to use several signal peptides such as LipB, WprA and NprE to express BgaB, and we obtained recombinant strains, such as WB600(pMALipB-bgaB),WB600(pMAWprA-bgaB) and WB600(pMANprE-bgaB). The results showed that, the expression levels of BgaB were quite different in the three recombinants: WB600(pMANprE-bgaB) expressedβ-galactosidase at the highest level among these three recombinants, reaching 2.091U/mL; WB600(pMAWprA-bgaB) expressed BgaB with a lower activity, only 0.328U/mL; however, the expression of BgaB in WB600(pMAWprA-bgaB) was not detected at all. These phenomenons indicate that signal peptides have a great influence on the expression of BgaB, even make the express of BgaB unsuccessful.In order to evaluate the influence of the hydrophobicity of BgaB on its secretion, We constructed a series of vectors by deletion some strong hydrophobic areas in BgaB(X=1,2,3,4,5), and then translated these vectors into WB600 to obatin several recombinants WB600(pMANprE-bgaB-X) (X=1,2,3,4,5). The results showed that secretion efficiency of BgaB was improved by the N terminal hydrophobic area delection, on the other hand, the deletion of other strong hydrophobic areas do not contribute to the scretion ,but reduce the secretion efficiency of BgaB .In addition, we also studied the influence of the BgaB length on its secretion.We constructed a series of vectors with different lengths of bgaB gene, namd as pMANprE-bgaB-Y(Y=A,B,C,D). Then transfered them into WB600 and obtained a series of recombinant strains WB600(pMANprE-bgaB-Y). The results showed that, under the condition of small length of BgaB ,the secretion efficiency improved with the decrease of the BgaB length.