| Galacto-oligosaccharide(GOS) are composed of 2-10 galactose and glucose unit linked by glycosidic bond. It is commercially produced from lactose through an enzymatic transgalactosylation reaction catalyzed by microbial β-D-galactosidase. GOS are known to be non-digestible oligosaccharide with low sweetness, low caloric values, low cariogenicity, and selectively promote the growth of beneficial bacteria in the colon. So it is a potential ingredient in food industry. The primary goal of this research was to increase the yield of GOS produced by β-D-galactosidase. Studies on the strain screening, β-D-galactosidase liquid fermentation, enzyme induction synthesis, purification, enzymatic properties, factors affecting GOS formation and separation of GOS were systematically carried out in this paper. The main results were as follows.A strain overproducing β-D-galactosidase was obtained out of 860 colonies screened by using 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside(X-gal) as indicator from mutagenized Kluyveromyces. fragilis cells with ethyl methanesulfonate(EMS) as mutagen. β-D-galactosidase produced by this mutant showed higher transgalactosylation activity compared with the wild-type strain, and it was coded Kluyveromyces fragilis LFS-8611. The maximum yield of GOS produced by K. fragilis LFS-8611 β-D-galactosidase was 29.6%, which was 2.8-fold higher than that produced by wild-type strain. The optimal culture medium for K. fragilis LFS-8611 β-D-galactosidase production was 12mg/mL lactose, 16mg/mL peptoneF403, and the optimal culture conditions were initial pH 7.5, rotation 200r/min, and temperature 25℃. The K. fragilis LFS-8611 β-D-galactosidase was inducible. Galactose and glucose were good inducers, and their induction coefficients were 2.8 and 1.9, respectively. The K. fragilis LFS-8611 β-D-galactosidase activity reached maximum value, 18.86 U/mL, with 10mg/mL galactose as inducer after 4h induction. Enzyme induction required the constant presence of inducer, since removal of inducer caused a rapid reduction in enzyme activity. Glucose neither excluded galactose and lactose from entering K. fragilis LFS-8611 cells, nor repressed β-D-galactosidase induction synthesis. Galactose had inductive effects on K. fragilis LFS-8611 cells growing at early, middle, or late log-phase, but induction coefficient was large for cells growing at early log-phase.The β-D-galactosidase from K. fragilis LFS-8611 was purified by two different chromatography methods. The enzyme was purified about 45.8 fold with a yield of 16% total activity by successive ammonium sulfate-fractionation, CM-Sepharose CL-6B, DEAE-Sepharose CL-6B, and Sephacryl S-200 column chromatography; and it was purified about 42 fold with a yield of 27% total activity by Hiprep?16/10 Source TM 30Q and Hioad26/60 Superdex medium-pressure liquid chromatography. When the purified enzyme samples obtained by the two different chromatography methods were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE), two single bands were observed for the two enzyme samples, respectively. which suggested that the two enzyme samples were almost pure. The molecular weight estimated for K. fragilis LFS-8611 β-D-galactosidase by sodium dodecanesulphonate polyacryl.amide gel electrophoresis(SDS-PAGE) and gel filtration on SepharoseCL-6B was 60000 and 68000, respectively.The optimum temperature and pH for purified K. fragilis β-D-galactosidase LFS-8611 were 45℃and 7.5, respectively. The enzyme was stable over a range of pH7.5-8.0 after treatment at 40℃ for 6h. The enzyme showed high stability at 25-40 ℃, and didn't lose its activity after 6h incubation at pH7.5. Pb2+, Cu2+, Zn2+, sodium dodecyl sulphate (SDS) and ethylenediaminetetraacetic acid (EDTA) had inhibitory effect on β-D-galactosidase activity. The Km and Vmax value of the enzyme for the synthetic substrate o-nitrophenyl-β-D-galactoside(ONPG) was 2.35 μmol/mL and 1.41 μmol/min, respectively, while that for natural substrate, lactose, was 0.39 μmol/mL and...
|
PDF Full Text Request