Functional Characterization Of Lactobacillus Sp.B164 β-galactosidase And The Effect Of ELP-system On Its Expression,Purification And Lactose Hydrolysis Efficiency

Backgroundβ-galactosidases are widely used industrially for the production of low-lactose milk by catalyzing the hydrolysis of lactose into glucose and galactose.The enzyme has other significant industrial and biotechnological applications including whey treatment,production of galacto-oligosaccharides and the bioconversion of lactose into products that can be easily converted to bioethanol.For this reason,techniques for expressing and purifyingβ-galactosidase have become an important subject which are currently under critical investigation.Research MethodIn this study,β-galactosidase gene from the bacteria Lactobacillus sp.B164 was separately modified with（1）a Linker-6x-His tag to form the recombinantβ-Gal-Linker-His（β-Gal-LH）and（2）Linker,Elastin-like polypeptide and 6x-His to form the recombinantβ-Gal-Linker-ELP-His（β-Gal-LEH）.Each recombinant gene was constructed into the plasmid p ET28a（+）and sub-cloned into the E.coli strain BL21（DE3）for protein expression.The expressedβ-Gal-LEH enzyme was purified utilizing two methods,mainly the inverse thermal cycling（ITC）and Ni-NTA resin column whereas theβ-Gal-LH was purified with only Ni-NTA resin column.The purification efficiency of the two methods and the biochemical properties of the purified enzymes were then evaluated.Results（1）A one round ITC at 1.75M（NH₄）₂SO₄ showed a higher purification efficiency forβ-Gal-LEH.A second round ITC was also performed but resulted in a reduced amount of purified enzyme with little to no difference with respect to purity compared to the 1^st round,indicating that a one round ITC was enough to get a highly purified protein.（2）The specific activity,K_m and K_cat/s-1 forβ-Gal-LEH andβ-Gal-LH were 177.92 U/mg,10.5m M,0.399 and 122.90 U/mg,15.7 m M,0.143 respectively indicating that ITC had a better purification efficiency resulting in a higher activity compared to Ni-NTA.The optimal temperature and p H were found to be 40°C and 7.5 for bothβ-Gal-LEH andβ-Gal-LH.（3）β-Gal-LEH left at 50℃ for 30 minutes maintained residual activity of～80%whereas the residual activity ofβ-Gal-LH left at 50℃for 30 minutes was～15%of the original activity.β-Gal-LEH andβ-Gal-LH stored at 25°C for 30 days showed a residual activity of～92%and～55%respectively.Taken together,β-Gal-LEH showed a better thermal and storage stability compared toβ-Gal-LH.In addition,the ELP tag does not change the secondary structure of the enzyme.Whereas Hg²⁺,Cd²⁺,Cu²⁺and Zn²⁺ions completely inhibited the activity ofβ-Gal-LH andβ-Gal-LEH,EDTA,urea and Ca²⁺did not have any significant effect on the enzyme activity;low concentrations of Mg²⁺and Mn²⁺ions however slightly improved enzyme activity.（4）An evaluation of the hydrolysis efficiency ofβ-Gal-LEH on lactose substrates revealed that the enzyme could produce 80.84,69.30 and 64.96 mg/d L of glucose in evaporated milk,skimmed milk and whey permeate respectively at 50°C.A comparative analysis of the hydrolytic efficiency betweenβ-Gal-LEH andβ-galactosidase from Kluyveromyces lactis and Aspergillus oryzae further revealed thatβ-Gal-LEH could efficiently catalyze the hydrolysis of lactose but may require a relatively longer time（27 h）when compared to K.lactis（3 h）but performed relatively better when compared to A.oryzae.ConclusionWe demonstrate by this study that an ELP-tagged protein has an improved purification potential and yields an enzyme with an improved catalytic activity compared with the traditional column affinity chromatography method.Moreover,utilization of ITC is more efficient,less costly and highly applicable for industrial hydrolysis of lactose.