Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24

[Display omitted] •β-galactosidase genes lacLM, lacL, and lacM were isolated from L. acidophilus MR-24.•The lacLM, lacL, and lacM genes were inserted into the pTZ57R vector for cloning.•Co-expressing lacL and lacM in E. coli produced protein with a 30-fold increase in activity.•Purified enzymes showed stability at 7 pH and 60 °C and 45 °C for native and expressed proteins. The current study characterized β-galactosidase-producing bacteria isolated from dairy products, in Lahore, Pakistan. Biochemical tests and the 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) hydrolysis test identified 50 Lactobacillus isolates. Fifteen isolates with blue color on X-gal plates, L. acidophilus MR-24 displayed the highest enzyme activity (905.15 U/l), characterized based on 16S rRNA gene sequencing. The enzymatic activity was enhanced 10-fold by using a Sephadex G-75 column. The highest enzyme production was obtained at pH 7.0 and a temperature of 37 °C. The β-galactosidase (lac M, L, and LM with molecular weight 951 bp, 1887 bp, and 2.8 kb respectively), was extracted from L. acidophilus MR-24. It was ligated to the pTZ57R cloning vector after Polymerase chain reaction (PCR) and agarose gel analysis. The confirmation of cloning was done via colony PCR and restriction digestion analysis. Sequencing data indicated that the enzyme consists of two overlapping regions; lac L and M encoding 70 kDa and 35 kDa protein respectively. The β-galactosidase shows significant homology with the gene from other Lactobacillus sp. Compared to crude enzymes, the co-expression of lac L and M in E. coli produced active proteins with a 30-fold increase in activity after purification via ion-exchange chromatography. The purified enzyme revealed its maximal activity at pH 7 while pH 3.0 and 9.0 showed minimal activity. The optimum temperature was 60 °C for native and 45 °C for expressed enzyme, remaining 40 % activity at 90 °C. The enzyme’s 3-dimensional (3D) structureshowed Domain N, A, and C. The L. acidophilus MR-24 strain, as a probiotic in dairy products, can provide benefits to individuals with lactose intolerance.