Session: General Poster:

β-galactosidase (EC 3.2.1.23), catalyzing the hydrolysis of β-1,4 or β-1,6 galactosyl bonds in disaccharides as well as transglycosylation, is a kind of glycoside hydrolase that plays a important role in dairy industry. In this study, a novel thermal stable β-galactosidase (lacZBa) was characterized from Bacillus aryabhattai GEL-09. It showed that the lacZBa encoding gene was composed of 1950 bp open reading frame (ORF), which encodes a protein containing 649 amino acid residues. Phylogeny and amino acid sequence analysis indicated that this enzyme belonged to family 42 of glycoside hydrolases. The lacZBa encoding gene was over-expressed in E. coli BL21(DE3) and the recombinant enzyme was purified to homogeneity. The molecular weight of the recombinant β-galactosidase was estimated to be about 75.4 kDa by SDS-PAGE. The recombinant β-galactosidase exhibited maximum activity at 45°C, and pH 6.0, and it maintained 92%, 96% and 47% relative activity at 40°C, 50°C and 55°C, respectively. The activity of enzyme can be activated when it was incubated at 45°C for several minutes. Moreover, the residue activity of the purified enzyme remained about 150% after being stored at 45°C for more than 100 h, and its half-life was 36 h at 50°C. In the presence of Co²⁺, Mn²⁺, Zn²⁺, Fe³⁺, Mg²⁺, Ca²⁺，the activity of the recombinant enzyme was enhanced, however, copper ionand EDTA had a significant inhibitory effect on enzyme activity. In addition, the kinetic analysis showed that the michaelis constant (K_m) value was 14.38 mM and the maximum rate of enzymatic reaction (V_m) value was 11.66 U/mg when oNPG was used as the substrate. In this study, a solution with a concentration of 5 g/L lactose was used to simulate the commercial milk, and the lactose substrate was completely hydrolyzed within 2 h with addition of 7 U/mL of pure enzyme at 45°C. These results suggested that the β-galactosidase identified in this study has potential applications in the dairy industry.

Shuyue Luan

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