Functional expression of microbial phytase genes in industrial host

Abstract

Cereals, legumes, and oilseed crops are grown in over 90% of the world''s harvested area. These crops serve as a major source of nutrients for humans and animals. An important constituent in these crops is phytic acid(myo-inositol hexaphosphate). the salt form, phytate, is the major storage form of phosphorus and accounts for more than 80% of the total phosphorus in creals and legumes. Phytase is an enzyme capable of hydrolyzing phytic acid to less-phosphorylated myo-inositol derivates. Monogastric animals, such as pig, poultry and fish, are not able to metabolized phytic acid, and therefore inorganic phosphate is added to their diets to safety the phosphorus requirement. This consequently contributes to phosphorus pollution problems in areas of intensive livestock production. Phytic acid also acts as an anti-nutritional agent in monogastric animals by chelating various metal ions needed by the animal, such as calcium, copper, and zinc. Therefore the enzymatic hydrolysis of phytic acid into less phosphorylated myo-inositol derivatives in the intestine of monogastric animals is desirable. Many attepts to enzymatically hydrolyze phytic acid have been made to improve the nutitional value of feed and to decrease the amount of phosphorus excreted by animals. So, this study was conducted to construct the recombinant phytase gene from Pseudomonas syringae MOK1 and Penicillium oxalicum PJ3 with commercial vectors in industrial hosts and to characterize the recombinant proteins. 1. Psudomonas syringae MOK1 genes with pET vector were transfored into E. coli BL21(DE3). 2. Transformed cells were induced by IPTG, after 6 h the cell extract were assayed phytase activity. The result of phytase activity was about 0.4 U/ml 3. Penicillium oxalicum PJ3 phytase gene with pPICZ¥áA vector were transfored into P. pastoris GS115 Mut+. 4. Recombinant phytase activity was increased steadily up to 120 h after the methanol induction and cell growth pattern was also similar to increase ratio of phytase activity. 5. The enzyme had a optimum pH of 4.5, and showed over 70% of the maximum activity in the pH range of 3.7-5.0. The optimum temperature for the enzyme activity was 55¡É, and showed over 70% of the maximum activity in the broad temperature range of 38-61¡É. 6. The specific activity of the purified enzyme was 306.62U/mg of protein at 55¡É and pH 4.5. 7. The Km and Vmax values determined by the Line-Weaver Burk plot were 0.37 mM and 526.3 U/mg respectively. 8. In SDS-PAGE analysis, the sample contains little minor contaminated proteins. Upon deglycosylation in vitro by Endo H, the molecular size of the expressed phytase by the control cells was reduced from 67kDa to 50kDa.