【作者】 张霞；

【导师】 袁其朋；

【作者基本信息】 北京化工大学 ， 化学工程与技术， 2015， 硕士

【摘要】 乳糖酶,学名为β-半乳糖苷酶,能够特异性的水解p-D-半乳糖苷键,催化乳糖水解,生成葡萄糖和半乳糖。乳糖酶的应用范围非常广泛,它能够水解牛奶及乳制品中的乳糖,减少乳糖不耐症的发生；可以用于改良乳制品；用于分析检测等。目前工业上生产的乳糖酶成本高,本研究利用基因工程技术,尝试在大肠杆菌和毕赤酵母中表达外源的乳糖酶,对乳糖酶的酶学性质进行研究,发酵条件进行优化,得到如下结论：1)分别成功构建了pETDuet-1-lac4, pMAL-c2x-lac4, pET22b（+）-lac4, pET28a（+）-lac4四种不同质粒,并对其进行诱导表达,其中pET22b（+）-lac4重组质粒的乳糖酶的酶活达到最高2.121 U.mL-1发酵液。2)测定了pET22b（+）-lac4的重组酶的酶学性质,乳糖酶的最适pH 7.0,最适宜温度为45℃,在45℃保存4h酶活依然在80%以上,K+对于乳糖酶酶活基本没有影响,Mn²⁺,Ca²⁺浓度为分别为1 5 mM,25 mM时乳糖酶的酶活达到最大,Zn²⁺对于乳糖酶的酶活具有抑制作用。3)对于大肠杆菌的发酵条件,诱导的条件进行了优化,在甘油浓度为4%,酵母粉3%,蛋白胨3%的条件进行发酵,IPTG诱导浓度为0.5 mM,在28℃条件下诱导21 h,检测到的乳糖酶的酶活达到37.38 U.mL-1发酵液。4)成功构建了毕赤酵母的分泌型重组质粒,但没有检测到乳糖酶的酶活。更多还原

【Abstract】 Lactase, also known as β-D-galactosidase, is capable of specifically hydroly-zing the β-D-galactoside bond. Lactase can catalyze hydrolysis of lactose to produce glucose and galactose. Lactase has wide range of applications, it is capable of hydrolyzing lactose in milk and dairy products, to reduce the incidence of lactose intolerance; it can be used to improve dairy quality; it can be used for the production of galactooligosaccharides; it can be used for analysis testing. Currently lactase high cost of industrial production, this study using genetic engineering techniques, try lactase expression in E. coli and exogenous Pichia, through a series of studies, the results are as following conclusions:（1） In this paper, four different plasmids pETDuet-1-lac4, pMAL-c2x-lac4, pET22b（+）-lac4, pET28a（+）-lac4 were constructed successfully which were expressed in E. coli. Lactase was expressed in E.coli.The enzyme activity of pET22b （+）-lac4 of is 2.121 U·mL-1.（2） Measure the enzymatic propertiesof the pET22b （+）-lac4 including the optimum temperature, pH, temperature stability et al.The most suitable pH is 7.0, optimum temperature is 45 ℃, it has good stability at 45 ℃, K+ had no effect for lactase activity, when the concentration of Ca²⁺ is 25 mM,lactase activity reaches the maximum. the concentration of Mn²⁺ is 15 mM, lactase activity reaches the maximum. Zn²⁺ has inhibitory effect on lactase enzyme activity（3） E. coli fermentation conditions were optimized.When the glycerol concentration is 4%, yeast extract is 3%, peptone is 3%, IPTG-induced concentration is 0.5mM, the temperature is 28℃ and ferment for 21h, lactase activity reached 37.38 U·mL-1.（4） Recombinant Pichia yeast plasmid were constructed successfully, but lactase is not detected.更多还原