Gene Engineering Technologyin The Production Of Lactase

Lactase, also known as ?-D-galactosidase, is capable of specifically hydroly-zing the ?-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.