| Summary: | 博士 === 靜宜大學 === 食品營養學系 === 103 === To obtain the phytase gene from Escherichia coli BL21 DE3, express in Pichia pastoris system and characterize their bioproperties, the gene encoding E. coli BL21 DE3 phytase was amplified by PCR. The sequence of phytase was confirmed and ligated into pGAPZaC expression vector. The plasmid (pGAPZaC-phyA) transformed into Pichia pastoris SMD1168H expression host. The recombinant phytase was expressed and secreted into broth by P. pastoris SMD1168H after 96 h cultivation and the activity was reached to 15.9 U/mL. The phytase was purified by nickel affinity chromatography and characterization of recombinant phytase. The recombinant protein with optimal pH and temperature at 5.0 and 50 oC, respectively. It was stable at pH 3.0-6.0 and 20-40 oC. The purified recombinant phytase was strongly inhibited by Cu2+, Fe2+, Hg2+, Fe3+, phenylmethyl sulfonylfluoride (PMSF) and N-tosyl-L-lysine chloromethyl ketone (TLCK), but activated by Ba2+, Mg2+, Ca2+, Sr2+. The purified recombinant phytase was sensitive to pepsin.
To optimize the phytase gene expression in Pichia pastoris system and characterize the recombinant phytase, a mature phytase cDNA of E. coli being altered according to the codons usage preference of Pichia pastoris was artificially synthesized. Pichia pastoris mutant with E. coli phytase optimized gene was cloned into expression vector of pGAPZaC. The phytase activity was 201.1 U/mL after 114 h fermentation in 5L fermentor. The recombinant phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The phytase had an optimal pH and temperature of 5.0 and 50 oC, respectively. It was stable at pH 3.0-8.0 and 25-40 oC. The purified recombinant phytase was resistant to trypsin and revealed higher affinity to calcium phytate than to other phosphate conjugates.
|
|---|
