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 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。
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 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( pichiapastoris )系统表达该基因进行了探索。
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 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。
So the lactase was suitable to whey at ph7 . 0 , but could n ' 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 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。
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 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 。
