| Summary: | 博士 === 國立臺灣大學 === 食品科技研究所 === 94 === β-Galactosidase produced by some microorganisms, in addition to catalyze the hydrolysis of lactose, exhibits a transgalactosylation activity (TGase activity) which can catalyze the formation of galactooligosaccharides (GOS), a prebiotics, from lactose. In this study, experiments were conducted to investigate the production of β-galactosidase which processes transgalactosylation activity from Bifidobacterium. In addition, purification and characterization of β-galactosidase from B. longum BCRC 15708 as well as the synthesis of GOS with this enzyme were performed.
β-Galactosidase production by Bifidobacterium strains, including B. longum BCRC 14634, 15708 as well as B6, B. breve BCRC 11846, B. bifidum BCRC 14615, B. adolescentis BCRC 14608, and B. infantis BCRC 14633, was first carried out in flask, with B. longum BCRC 15708 showing the highest production of β-galactosidase with the highest specific activity. Further study with B. longum BCRC 15708, it was first found that lactose and yeast extract, respectively, were the best carbon source and nitrogen source for β-galactosidase production. After 16 hrs cultivation, a maximum β-galactosidase activity of ca 18.6 U/ml was obtained in medium containing 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4•7H2O, 0.03% L-cysteine, and with the initial pH value and temperature controlled at 6.5 and 37℃, respectively.
β-Galactosidase from B. longum BCRC 15708 was purified by procedures including Q Fast-Flow chromatography and Superose 6 HR gel chromatography. These steps resulted in a purification of 15.7-fold, a yield of 29.3% and a specific activity of 168.6 U mg-1 protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate, the pH and temperature optimum of the purified β-galactosidase were 7.0 and 50℃, respectively. The Km and Vmax were 0.85 mM and 70.67 U/mg, respectively. Monovalent cations (Na+ and K+) stimulated the enzyme up to 10-fold, whereas Fe3+ and bivalent cations (Fe2+, Co2+, Cu2+, Ca2+, Zn2+, Mn2+, and Mg2+) in the concentration of 1 mM inhibit β-galactosidase activity. Furthermore, galactose, lactose, and fructose would also inhibit the enzyme activity.
B. longum BCRC 15708 grown in a jar fermenter showed that inoculum size, cultivation temperature, the pH of medium and agitation speed all would affect β-galactosidase production. A maximum β-galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ml were achieved after 10 hr of fermentation with 20% inoculum of the test organism into a medium containing 4% lactose and 3.5% yeast extract, and with the pH of medium, cultivation temperature and agitation speed controlled at 6.5, 37℃ and 100 rpm, respectively.
Two types of GOS, tri- and tetrasaccharides, were formed by transgalactosylation catalyzed by β-galactosidase from B. longum BCRC 15708, while trisaccharides were the major type of GOS formed. A maximum yield of 30.1% (w/w) GOS could be achieved from 40% lactose solution at 45℃ and pH 6.8 when the lactose conversion was 57.8%. The corresponding productivity of GOS was 13.9 g/l•h. Moreover, when more than 15% of galactose or glucose was added to reaction mixture containing 40% lactose, transgalactosylation reaction was markedly inhibited by 17.3-35.8%.
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