Study On Fermentation, Separation, And Characteristics Of β-D-galactosidase

Beta-D-galactosidase (EC3.2.1.23) is a kind of hydrolytic enzyme, catalyses the Galactosyltransferase reaction to produce Galacto-oligosaccharide (GOS). The food industry uses this enzyme to catalyse hydrolysis of lactose and to produce low lactose dairy products. So,β-D-galactosidase has been widely used in the dairy industry.Beta-D-galactosidase,commonly named lactase which not only can hydrolyze theβ-1,4-galactosidic linkage but also has transglycosylation activity, is a very important glycosidase and plays an important role in food, medicine, analytical chemistry and other fields. In recent years, with the development of glycobiology,β-D-galactosidase has become to be an important tool for saccharide synthesis.Our laboratory selected a Kluyveromyces marxianus SK16.001,β-D-galactosidase produced by the strain has a high activity of transglycosylation, and has the industrial application potential, so it is necessary to know the optimum condtions of the fermentation, and also separate and purify this enzyme, to study its enzymatic properties.The optimal culture medium for Kluyveromyces marxianus SK16.001β-D-galactosidase production was 20g/L lactose, 20g/L peptone, 10g/L yeast extract,NaH₂PO₄ 0.04% and the optimal culture conditions were initial pH 6.5, temperature 30℃and rotation 150r/min. Under the optimal culture conditions, the activity ofβ-D-galactosidase was 13.24U/mL.The enzyme purification was done by means of ammonium sulfate faction, dialysis, DEAE ion-exchange chromatography, Superdex G-75 gel filtration . The purified enzyme identified by SDS-PAGE indicated that a single protein band was obtained. Its molecular weight is about 63315 Da.Then we studied the enzyme characterization of the purifiedβ-D-galactosidase. It exhibited optimum activity at 35℃and pH7.0. The enzyme activity was partly inhibited by Mg²⁺, Co²⁺, and was strongly inactivated by Ca²⁺, Fe²⁺, Cu²⁺, but it was activated by Mncf.