Cloning, Expression Of Thermostable Lactase Gene In E. Coli And Enzymatic Properties

It has been estimated that about70%to95%of people inhabitant suffer from either lactose maldigestion or intolerance. Lactase can be used for treatment of "lactose intolerance" and production of low-lactose milk, whey and other dairy. So lactase is valuable in both theory and practice.The lactase gene from Bacillus coagulans T242was researched in this thesis. The cloned gene was expressed in E.coli and the properties of lactase was determinated.Specific primers were designed according to the sequence of the bete-galactosidase genome from Bacillus coagulans36D1and the sequence of genomic DNA. The gal gene was amplified by PCR with the Bacillus coagulans T242genomic DNA as a template, and subsequently cloned, then ligated to the pET32-a(+) vector, named pET32-gal. The recombinant plasmid was transformed into E.coli HST08, screened positive transformants. Then the recombinant plasmid was trasformed into E.coli BL21. The positive transformants were cultured at37℃in LB containing100u g/mL Amp to an OD600of0.6, then were induced at25℃or37℃for four hours with IPTG,which final concentration was1mmol/L. The cells were harvested after4h of cultivation by centrifugation. The cells were disrupted by sonication. SDS-PAGE analysis showed that numerous target protein was detected,which demonstrated the expression was inclusion. Expresstion plasmid pET32-gal and the molecular chaperone pGro7plasmid transformed E.coli BL21to realized the soluble expression of lactase gene. SDS-PAGE analysis showed that whey of disrupted cells contained target protein. To further optimizate the induced culture conditions, pET32-gal/pGro7/BL21was cultured at the temerperature of37℃for4h when the IPTG concentration was0.6mmol/L. Lactase activity was maximal, up to6.791U/mL, which was5times higher than the original strain.Preparing the lactase solution expression in E.coli, we deteminated the optimum pH, pH stability, optimum reaction tempreture, thermal stability, effect of metal ions and chemical reagent, Km and Vmax. Its optimum pH was6.8and good pH stability from6.0to9.0. So the lactase was suitable to whey at pH7.0. The optimu temperature was50℃and good tempreture stability from40℃to50℃, but at60℃, the lactase activity fallen rapidly. So it couldn’t react at high tempreture. The metals Mg2+, Ca2+and SDS could prominently activate the enzyme, Cu2+, Pb2+, Zn2+, Mn2+could slightly inhibit the activity, but the Fe2+would strongly inhibit the activity. The K+, EDTA had no effect to this lactase. At50℃and pH6.6, the Km was7.60mmol/L and the Vmax was128.21nmol/min. In summary, the high activity of the enzyme in ONPG hydrolysis at pH of6.8and in the presence of Ca2+ions suggests potential usefulness of this enzyme in the hydrolysis of milk lactose.