关键词: β-半乳糖苷酶；低聚半乳糖；转糖基反应；开菲尔乳杆菌；酶学特性

Abstract: In order to provide a novel enzyme source for the high-efficiency synthesis of galacto-oligosaccharides (GOS), novel lactic acid bacteria (LAB) strains capable of producing β-galactosidase with high transglycosylation activity were screened from camel milk in the present study. The primary screening was performed using MRS plates with lactose as the carbon source supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). The secondary screening was carried out through thin layer chromatographic (TLC) analysis of the products of transglycosylation catalyzed by microbial β-galactosidase. The conditions for enzyme production and transglycosylation reaction were optimized by one-factor-at-a-time method. The β-galactosidase was partially purified by fractional precipitation with ammonium sulfate and its enzymatic properties were characterized preliminarily. Twenty LAB strains producing β-galactosidase with transglycosylation activity were obtained, among which strain L6 produced β-galactosidase with the highest transglycosylation activity and it was identified as Lactobacillus kefiri by physiological, biochemical and molecular biological approaches. The optimal culture conditions that provided maximum activity level of (3.81 ± 0.02) U/mL were determined as follows: concentration of carbon source (lactose) 2 g/100 mL, concentration of nitrogen source (peptone, beef extract, and yeast extract) 1 g/100 mL, initial pH 5.5, and culture at 37 ℃ for 20 h. The β-galactosidase produced by L6 had a broad reactive temperature range of 45–70 ℃ with more than 50% activity being retained. After 4 h reaction at 65 ℃ and pH 7.0 with 45 g/100 mL of lactose as the substrate, the β-galactosidase could produce GOS-2, GOS-3 and other GOS with a yield of 13.51%, 13.85% and 4.15% (m/m), respectively.

Key words: β-galactosidase; galacto-oligosaccharides; transglycosylation activity; Lactobacillus kefiri; enzymatic properties