Expression Of Lactase From Kluyveromyces Lactis In E.coli And Study On Enzymatic Properties

β-D-galactoside(EC 3.2.1.23) is usually named lactase which can hydrolyze lactose into galactose and glucose.It can transfer galactoside.Lactase is mainly used in producing low lactase dairy products and can be used for treatment of "lactose intolerance".It plays an important part in immunity,detection and disease diagnosis.The lactase gene Lac4 of Kluyveromyces Lactise was expressed in Escherichia coli and analyzed the characterization of the recombinant lactase in this research.The main results were as follows:1.The expression of lactase gene in E.coli.The recombinant plasmid pET-28a-lac4 was transformed into E.coli BL21.The expression of recombinant protein was induced in E.coli with IPTG and detemined by SDS-PAGE after sonication of bacterial pellet. Recombinant lactase existed at supernatant and inclusion bodies.The recombinant lactase was purified with Ni-NTA affinity chromatography and determined protein concentration by Bradford Assay.The results indicated that the concentration of purified lactase was 80.56μg/mL,enzyme activity was 20.59±0.19 U/mL,and specific activity for 255.55±2.32 U/mg respectively.2.Analysis of lactase characterization.A single band was showed on SDS-PAGE after purifying lactase solution using Ni-NTA column.Recombinant lactase is neutral enzyme and its molecular weight is about 122 kDa.Its optimum reaction pH is 7.0,and has good stability within the pH range from 4.0 to 10.0.The optimum reaction temperature is 43℃,it has better thermal stability at 37℃,its half-life is 30min;Ca²⁺,Zn²⁺,Fe²⁺,Co²⁺,Mg²⁺,Man²⁺ have good activation on lactase respectively,but Cu²⁺ has a strong inhibitory effect on the enzyme.Its K_m is 3.49 mmol/L and V_max is 4.22 mmol/min.mg,determined using different concentrations of ONPG as substrate and double-reciprocal plot method at 43℃,pH 7.0 conditions.3.Optimization of fermentation conditions of lactase genetic engineering strain. Using orthogonal design test to optimize the formula for engineering strain medium and induced expression conditions.The results showed that the best formula for fermentation medium was that yeast powder was 3.0%of the total weight of the medium,and typtone 2.5%,glucose 2.5%,NaCl 1.0%respectively;the best conditions for culturing and induction were as follows:induced temperature was 28℃,liquid volume was 10%, inoculum was 1%,IPTG concentration was 0.15 mmol/L,and induced for 4.5 h.Activity of lactase can reached 116.69 U/ml under the best inducing condition.