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 ion and EDTA had a significant inhibitory effect on enzyme activity. In addition, the kinetic analysis showed that the michaelis constant (Km) value was 14.38 mM and the maximum rate of enzymatic reaction (Vm) 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.

Cloning and expression of a novel thermal-activated β-galactosidase from Bacillus aryabhattai Gel-09

ACS Spring Meeting 2021

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