P160: Overexpression of α-fucosidase in Lactobacillus casei BL23 using nisin controlled expression system

In order to utilize fucosylated human milk oligosaccharides (HMO), Lactobacillus casei BL23 possesses putative three fucosidases, implicating GH29 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Thus, to link genetic structure with an encoded enzyme’s substrate, and to determine the extent to which a given substrate induces expression, the expression profiles of all three BL23 α-L-fucosidases were examined. According to qRT-PCR and phylogenetic analyses, Lcabl_28270 was considered the major gene responsible for the utility of fucosylated HMO. In this work the Lcabl_28270 encoding α-L-fucosidases from L. casei BL23 were cloned into pET system and expressed in E. coli BL21(DE3) Star and also cloned into nisin-controlled expression (NICE) vector and expressed in L. casei BL23. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 570 U/mg and the gel permeation chromatography analysis showed that the purified enzyme exists in tetramer in solution. The pH and temperature optima were pH 6.0 and 50°C, respectively. The enzyme hydrolyzed α1,2-fucosidic linkage but not α1,3 or α1,4-linkage of fucosylated HMO. Finally, the successful over-expression of α-L-fucosidase in L. casei BL23 was confirmed by enzyme activity assays and, those levels gradually increased up to 3.73 U/mL of culture broth after 24 h, followed by a decrease thereafter. On the contrary, wild BL23 showed little enzyme activity during the cultivation period.