Cloning, expression and characterization of a heterodimeric beta-galactosidase from Lactobacillus acidophilus ATCC 4356

Abstract:

Heterodimeric beta-galactosidase of Lactobacillus acidophilus belongs to glycoside hydrolase family 2, encoded by two overlapping and translational coupling genes, lacL and lacM. The lacL and lacM genes of the sequenced strain L. acidophilus NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively. [objective] To clone, overexpress and characterize the enzyme in Escherichia coli. [Methods] We cloned the fragment (2834 bp) containing ribose-binding site (RBS) and coding regions of the lacLM genes from L. acidophilus ATCC 4356 into expression vector pQE31. RBS and HIS-Tag of pQE31 were substituted by inserted fragment. Recombinant plasmid was electrotransformed into E. coli JM109. Expression product was purified by ammonium sulphate fractionation, an-ion-exchange, affinity chromatography and gel permeation. Native molecular mass of homogenous enzyme was measured by gel permeation, and beta-galactosidase activity was determined by using o-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. [Results] Overexpression of the soluble enzyme in E. coli was achieved. Amino acid residue 512 of re-combinant LacL was different from that of L. acidophilus NCFM. Homogenous enzyme was obtained by purification. The homogenous enzyme had a specific activity of 226 U/mg protein, a native molecular mass of 96.3±4.6 kDa, an optimum temperature at 49℃ and an optimum pH of 7. The Km and Vmax values of the enzyme were 2.18±0.12 mmol/L, 273±5 U/mg protein respectively.

Key words:Lactobacillus acidophilus; beta-galactosidase; clone; expression; purification; Km value