An elastin-like polypeptide (ELP) sequence fused with Lactobacillus sp. B164 β-galactosidase modified with 6x-Histidine (β-Gal-LH) to produce recombinant β-Gal-Linker-ELP-His (β-Gal-LEH) was expressed in E. coli and purified via inverse thermal cycling (ITC) and nickel-nitrilotriacetic acid (Ni-NTA) resin. The β-galactosidase integrated with ELP-system showed an improved purification at 1.75 M (NH4)2SO4 after 1 round ITC (95.66% recovery rate and 13.04 purification fold) with better enzyme activity parameters compared to Ni-NTA. The enzyme maintained an optimal temperature (40 °C) and pH (7.5) for both β-Gal-LEH and β-Gal-LH. The results further showed that the ELP-fusion system improved the enzyme's thermal and storage stability. Moreover, the enzyme secondary structure was not changed by ELP-tag. Enzyme activity was completely inactivated by Hg2+, Cd2+ and Cu2+, unaffected by Ca2+, EDTA and urea, but partially activated by Mn2+ at lower concentration. Compared to commercial β-galactosidases, β-Gal-LEH exhibited similar biocatalytic efficiency on lactose and could potentially catalyze transgalactosylation.
Keywords: Elastin-like polypeptide; Hydrolysis; Purification; β-galactosidase.
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