Cloning Of GalU Gene From E.Coli Into Lactococcus Lactis And The Impact On The Exopolysaccharide Production

Some of lactic acid bacteria (LAB) produce exopolysaccharide (EPS). The EPShas great potential applications in food and dairy products for its promising biologicaland rheological properties. It also has many physiological activities, such asimproveing mucous adhesion, antitumor, antiulcer, immunostimulatory effect etc. Inaddition, LAB possess GRAS (generally regarded as safe) status, which can ensurefood safety and be applied to food directly. However, the production of EPS by LABare unstable. The yield and composition of EPS could be affected by many factors,such as strains, culture conditions, carbon and nitrogen sources, and biosynthesisregulation. In recent years, methods to increase EPS production have been widelystudied, including optimizing media and fermentation conditions, and molecularmethods to overexpression of enzymes involving EPS biosynthesis, clone and expressof the complete eps gene cluster into other microorganisms etc.In the present study, a strain of Lactococcus lactis that producesexopolysaccharide was screened. The galU gene from E. coli was inserted intoLactococcus lactis, and the effect on the exopolysaccharide production wereinspected. The main results were as follows:1. The viscosity of fermented liquor and the yield of EPS poduced by lactic acidbacteria strains were determined to screen the EPS-producing bacteria. The highestone of EPS yield was Lactococus lactis L18. The viscosity was 349.42 mPa.s and theyield of exopolysaccharide was 716.05 mg/L detected by phenol-sulphuric acidmethod. 8% trichloracetic acid was used to deproteinize in the extraction of EPS fromfermented broth.2. The clone vector pMD18-T-galU were constructed. The plasmid harbouringgalU gene from E. coli was confirmed with in-situ PCR, restriction mapping, andsequence analysis. In comparison to the DNA sequence of E. coli, there was adifference of only 3 bases, and the protein sequence was the same.3. The expression vector pNZ8048-galU were constructed based on pNZ8048and pMD18-T-galU, and transformed into L. lactis L18. The recombinant cell was analyzed by PCR, enzymatic digestion, and sequence analysis. The results showedthat the construction was correct, including the size, the sequence, the position andthe direction. The SDS-PAGE analysis of expression products indicated that thehighest yield of galU (33.0 kD)was obtained when induction with 20 ng/mL nisin, 3hs, about 18.6 percent of all intracelluar protein.4. The EPS production of L. lactis L18 and recombinant L. lactis L18/pNZ8048-galU were compared to test the effect of the inserted gene, which mayreflect (?)he activity of UGPase. Different carbon sources, pH, temperature andcultivation time were tested for EPS production. Under the conditions of MRSmediu(?)ading glucose and lactose (20:20 g/L), pH6.5, 30℃, 1489.65 mg/L EPS wasproduced by L. lactis L18/pNZ8048-galU after 26h, while 848.93 mg/L EPS wasproduced by L. lactis L18 after 28h. Therefore, the EPS yield from L. lactisL18/pNZ8048-galU was 1.75 times to L. lactis L18.