摘要
β-半乳糖苷酶(B-D-半乳糖苷半乳糖水解酶，β-D-galactoside galactohydrolase，EC3.2.1.23)通常被称为乳糖酶(Lactasc)，能将乳糖水解为半乳糖和葡萄糖，并具有半乳糖苷的转移作用。乳糖酶主要用于改良乳制品和生产低聚半乳糖，还可用于蛋白质合成与遗传因子等方面的研究，具有重要的实践价值和理论意义。目前，工业生产中使用的乳糖酶主要来源于乳酸克鲁维斯酵母菌、黑曲霉和米曲霉。
本论文从一株乳酸克鲁维斯酵母菌中克隆获得乳糖酶基因，在大肠杆菌中进行表达并测定其酶学性质，同时也对利用巴斯德毕赤酵母(Pichia Pastoris)系统表达该基因进行了探索。
研究内容及结果如下：
1．乳糖酶基因的克隆及原核表达
以乳酸克鲁维斯酵母(Kluyveromyces lactis)菌株k538的基因组DNA为模板，设计引物，利用PCR获得乳糖酶基因Klac，经DNA测序验证，得到克隆T1549。乳糖酶基因插入到表达载体pET-30a(+)上构建重组表达质粒pETlac4，将pEFlac4电击转化到大肠杆菌BL21中，分别研究了阳性转化子在28℃、37℃下经IPTG诱导3h后的蛋白表达情况。表达产物的SDS-PAGE分析和酶活性测定表明，Klac基因在大肠杆菌中获得活性表达，其中低温条件28℃下的表达水平明显高于37℃，28℃诱导的细胞经超声波破碎所测酶活力为0.475U/mL，37℃仅为0.134U/mL。
2．原核表达乳糖酶的酶学性质测定
制备原核表达乳糖酶的原酶液。以ONPG为底物，分别测定了其最适pH值及pH稳定性、最适温度及热稳定性、金属离子和化学试剂的影响以及K_m值和V_(max)值。该酶最适pH值为5.8和6.4～7.5，属中性酶，碱性条件稳定，酸性条件不稳定；最适反应温度45℃～52℃，热稳定性较差，不适于高温反应；Fe~(2+)、Mn~(2+)、Co~(2+)、Mg~(2+)和Zn~(2+)对乳糖酶有明显的激活作用，且该酶有一定的金属离子依赖性，Cu~(2+)具有很强的抑制作用；37℃、pH6.6条件下K_m=1.143 mmol/L，V_(max)=142.857nmol/min。
3．乳糖酶基因Klac在巴斯德毕赤酵母中表达
将Klac基因插入到载体pPIC9上构建重组表达质粒PIC9154983，电击转化巴斯德毕赤酵母受体菌GS115，经筛选和PCR鉴定，1#、8#和24#为重组菌株，Klac基因整合到GS115基因组中。三个重组菌株经甲醇诱导培养未检测到分泌的乳糖酶，但胞内均检测到有活性的乳糖酶，分别为0.1392U/mL、0.3702U/mL和0.2214U/mL，说明乳糖酶基因Klac在巴斯德毕赤酵母中获得表达。
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+, can
prominently 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.
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