Cloning And Expression Of The Lactase Gene From Kluyveromyces Lactis And Analysis Of Lactase Properties

β-D-galactoside galactohydrolase (EC3.2.1.23) is usually named lactase which can hydrolyze lactose into galactose and glucose, and also can transfer galactoside. Lactase is mainly used in improving dairy products and producing oligo-galactose, as well as in the research related to protein synthesis and heredity factor. So lactase is valuable in theory and practice. At present, most of lactases used in industry production come from Kluyveromyces, Aspergillus niger and Aspergillus oryzae.The lactase gene from Kluyveromyces lactis was researched in the thesis. The cloned gene was expressed in E.coli and the properties of lactase was determinated. In addition, we studied the expression of lactase gene in the methylotrophic yeast Pichia pastoris.The main experiments and results were as follows:1. Cloning and expression in E.coli of lactase.Specific primers were designed according to the sequence of the beta-galactosidase gene from Kluyveromyces lactis. Klac gene was amplified by PCR and subsequently cloned. Klac gene was ligated to the pET-30a(+) vector for expression.Then the recombinant plasmid pETlac4 was transformed into E.coli BL21.The positive transformants were induced at different temperature for three hours by IPTG with the final concentration of lmM. SDS-PAGE analysis and lactase activity assay showed that Klac gene was expressed in E.coli, and that the expression level at 28℃ was much higher than that at 37 ℃. The former was 0.475U/mL, and the later was 0.134U/mL.2. Determination of the properties of lactase expressed in E.coli.Preparing the lactase solution expressed in E.coli, we determinated the optimum pH, pH stability, optimum reaction tempreture, thermal stability, effect of metal ions and chemical reagent, Km and Vmax Its optimum pH was 5.8 and 6.4 to 7.5, and the optimum tempreture was 45℃ to 52℃. It had better basicity-tolerance and worse acidity-tolerance. So the lactase was suitable to whey at pH7.0, but couldn't react at high tempreture. The metals, Fe2+, Mn2*, Ca2+, Mg2+ and Zn2+, canprominently improve lactase activity, but Cu2+ would strongly inhibit the activity. According to the effect of EDTA, we concluded that the lactase depends on metal ions in a certain extent. At 37℃ and pH6.6, the Km was 1.143mmol/L and the Vmax was 142.857nmol/min.3. Expression of Klac gene in Pichia pastoris.Klac gene was inserted into the vector pPIC9 to construct recombinant plasmid P1C9154983, and then PIC9154983 was transformed by electricity pulse into GS115, Pichia pastoris acceptor. After screening, we obtained recombinant yeast 1#, 8# and 24#, that is, Klac gene was integrated into the genome DNA of GS115. By detecting the lactase activity,we knew that the Klac gene was expressed in vivo of Pichia pastoris,but the lactase was not secreted.