| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 100 === Large amount of phytase have been consumed in feed industry, especially for 6-p phytase with activity optimal at acidic pH. This study aimed to clone E. coli phytase and express in Pichia pastoris. A mature phytase cDNA of E. coli being altered according to the codons usage preference of Pichia pastoris was artificially synthesized and cloned into expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/ml after 72 h high cell-density fermentation. The recombinant phytase was purified to electrophoretical homogeneity after Ultrafiltration and nickel affinity chromatography. At this stage, the yield, purification fold, and specific activity were 69.0%, 34.7 folds and 42.6 kU/mg, respectively. The recombinant phytase were deglycosylated to produce a homogeneous 46kDa protein. The optimal temperature and pH were at 50oC and 4.0-6.0, respectively. However, it was resistant to trypsin and stable at pH 3.0-8.0 and 20-40oC. Cu2+, Zn2+, Hg2+, Fe2+, Fe3+, phenylmethyl sulfonylfluoride (PMSF) and N-tosyl-L-lysine chloromethyl ketone (TLCK) strongly inhibited, while Mg2+, Ca2+, Sr2+, Ba2+, glutathione (GSH), ethylene diamine tetraacetic acid (EDTA) and N-ethylmaleimide (NEM) activated its activity. The purified recombinant phytase revealed higher affinity to calcium phytate than to other phosphate conjugates. Freeze-dried crud phytase was very stable during 6 week storage at room temperature, 4oC and -20oC.
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