Purification Of Pectinase And Display Of Carcinoembryonic Antigen On The Cell Surface Of Lactic Acid Bactiria

1. Screening of bacteria that producing alkaline pectinase from tobacco leavesA bacterial strain with highly pectinase was isolated from tobacco leaves by determining the pectinase activity in different time and pH. This bacterium was primarily identified as Bacillus subtilis based on16S rDNA gene sequence analysis. The highest pectinase activity of the strain was obtained in16hours after fermented. Because the optimal pH is10, the pectinase produced by Bacillus subtilis P6is alkaline pectinase. The composition enzymes were determined. Bacillus subtilis P6was found that the amylase activity is45U/mL.2. Optimization of pectinase-producing medium and preliminary separation of alkaline pectinaseBy optimizating the N source, C source and inorganic salts, the medium composed with1%(v/v) bean cake,10%(v/v) malt juice and0.3%(v/v) CaCl2can induce the most production of alkaline pectinase. Under the optimal cultural conditions, the maximum pectinase activity reached690U/mL, the highest enzyme activity reported in literatures from our knowledge. This strain exhibited the potential application in the textile industry. To purify the pectinase from the cultures of this bacterium, a series of biochemical technologies such as ion exchange was applied. An active fraction which was dominant in the pectinase activity was obtained. And by the analysis of SDS-PAGE, the weight of pectinase was about44kD.3. Oral Vaccines by novel surface display systems of Lactic Acid Bacteria to display carcinoembryonic antigenLactic acid bacteria (LAB) are Gram positive bacteria that can ferment soluble carbohydrate. They are considered to be safe bacteria with a GRAS (generally regarded as safe) status. The studies on LAB as live vaccine vehicles for the expression of heterologous proteins or antigens have gained great interest recently. Lactococcus lactis, an important species of LAB, possesses many properties that make it an ideal candidate for expressing and displaying heterologous proteins on its cell surface. Here, the C-terminal sequence (1csB) of S-layer protein (S1pB) of Lactobacillus crispatus K2-4-3was cloned as an anchor protein for construction the target expression system. Carcinoembryonic antigen (CEA) was displayed successfully on the surface of L. lactis NZ9000, which was verified by SDS-PAGE and Western blotting. To determine whether this LAB strains expressing CEA intracellularly or at the surface of the bacterium could evoke an immune response, mice were immunized by administration of live recombinant bacteria by the oral route. By the analysis of ELISA and MTT, the recombination strain displaying CEA on the surface can induce significant antibody response of intestinal washings, but can not induce IgG antibody response in serum.