Abstract: A strain, named ZX-5, with the ability to produce a high yield of β-D-galactosidase was isolated from traditional Tibetan kefir, and identified as Kluyveromyces marxianus by DNA internal transcribed spacer (ITS) sequence analysis. We found 2.0% galactose and 1.0% tryptone to be the optimal carbon and nitrogen sources, respectively. The optimal fermentation conditions were determined as follows: temperature 30 ℃, initial medium pH 6.5, medium volume 30%, shaking speed 100 r/min, inoculum size 2.0% and fermentation time 36 h. The activity of β-D-galactosidase produced under these conditions was 2.60 U/mL. Furthermore, the crude enzyme solution was purified by ammonium sulfate fractionation and DEAE-Sepharose fast flow ion-exchange chromatography to a specific activity of 157.35 U/mg. The purified β-D-galactosidase exhibited the maximum catalytic activity at 35 ℃ and pH 6.0, and had good stability at 20–40 ℃ and pH 5.0–7.0. Mn2+ could promote the enzyme activity. When the β-D-galactosidase produced by strain ZX-5 was used to decompose lactose and produce galacto-oligosaccharide under the conditions of 35 ℃, lactose concentration of 60 g/100 mL and enzyme concentration of 1.0 U/mL, the hydrolysis rate of lactose was 68.34% (after 50 h) and the yield of galacto-oligosaccharide was 34.70% (after 40 h), showing good potential application prospects.

Key words: Tibetan kefir, β-D-galactosidase, fermentation conditions, isolation and purification, enzyme characterization, galacto-oligosaccharide