Phytase Appa NR Expression In Kluyveromyces Lactis And Characterization Of The Enzyme

Phytic acid is inositol-6-phosphate which exists widely in plant tissues and food products, its more than80%of the total phosphorus content in cereals and legumes, it is the main form of phosphorus in the plant feed. Phytic acid often chelating the mineral elements Ca2+、Mg2+、 Zn2+、Fe2+etc. The forming complex reduce the absorption and utilization of mineral elements and protein. Metal ions and protein are required by humans and animals.Phytase is found primarily in plant seeds and microbes, it can decompose phytic acid to inositol and phosphoric acid, removing the complexation of phytic acid and enzyme inhibition, thereby enhancing the nutritional value and bioavailability of the food and feed. But also the stomach of monogastric animals and intestinal lack of phytase, so the phytate with feces causing environmental pollution.Phytic acid hydrolysis products have very important nutritional value and medicinal value, development and utilization of phytase in the food industry、feed industry and livestock has important implications. Phytase produced by microbes have a good development prospects, but the level of the natural strains producing enzymes is very low, and its some properties can not be entirely suitable for feed processing, food and pharmaceutical production requirements, thus limiting the popularization and application of phytase.In this study we clone the phytase gene appA NR derived from E.coli K12into the expression vector pKLAC2of Kluyveromyces lactic yeast GG799strains, the recombinant expression vector pKLAC2-appA NR linearized with Sac Ⅱ and transformed into Kluyveromyces lactic yeast by chemical method.Transformants were obtained by plating on YCB selective medium plates and incubated at28℃for72hours.Recombinant strains were inoculated to the induced medium of the YPGal, after4days we detect the expression product by SDS-PAGE gel electrophoresis, observing a specific band about60-65kDa. We analysis the enzymatic properties of phytase appA NR showed that the optimum reaction temperature is55℃, between45℃-60℃, the activity maintained at above75%.The activity is still retained more than84%after incubating10min at90℃, so it has good thermal stabilitythe.Optimum pH of5.5, it has a strong enzyme activity in the pH2.0-6.5of the acidic pH range. The phytase appA NR of Kluyveromyces lactic yeast expression has a strong tolerance to pepsin and trypsin, the residual activity is87.3%and79.5%after120min with pepsin and trypsin.