Expression Of Neutral Phytase Gene From Bacillus Subtilis WHNB02 In Bacillus Megaterium

The neutral phytase gene which come from Bacillus subtilis WHNB02(GenBank Access No. AF220075) is the target gene in this research.In order to remove the signal peptide and insert SacI and BamHI sites at the 5' and 3' ends,designing primers which have the two restriction'enzyme sites after the signal peptide.The single amplified PCR fragment was about 1.1Kb.It's the target expression fragment.The pcr product was ligated with the Tvector pMD18T vector after it has been recycled and pured.Transform the ligation product to E.coli JM109.After pcr,restriction enzyme cutting and DNA sequencing,The positive transformants were detected.Extract the plasmid,cutting the plasmid and expression vector pHIS1525 with restriction enzyme SacI and BamHI,then ligate the plasmid and expression vector pHIS1525 dealed with restriction enzyme.Transform the ligation product to E.coli DH5α,and extract plasmid after getting the positive transformants.Transform the recombinant plasmid to Bacillus megaterium WH320 by electorporation.The positive transformants were screened on the LB plate(10μg/mL tetracycline added).The effective expression of the gene in the B.megaterium and the production which have the phytase activity were proved by measuring the phytase activity. SDS-PAGE showed that The molecular weight is about 55KDa.The neutral phrase haven't been put into market as commodity because of the lack of research and low output of the enzyme.This research make the neutral phytase express in the Bacillus megaterium,and improve its output effectively.The research has important academic value and application foreground.It's a good foundation of the neutral phytase being put into market as commodity.Farther more,the culture conditions were studied.The results show that:the seed age is 16h,the inoculation rate is 5%,the induced concentration is 0.2%,the induction time is 9 hours.Under the optimal culture condition,the enzyme activity of phytase in supernatant can come to 33.0U/mL which is about 165 times higher than the activity of the initial strain Bacillus subtilis WHNB02.The phytase activity reach 45.3 U/mL after 9 hours'fed-batch fermenfation.