Cloning, Expression, Purification And Determine Bioactivity Of A New Phytase Gene

Phytase was the generic name of enzyme which catalyze the hydrolysis of phytate into myo-inositol and phosphate. On one hand phytase could enhance the mono-stomach animal absorbing phosphorus and increase its utilization rate in animal feed about 50% .On the other hand it could degrade the product phytate binded by some multi-metal ions and proteins then accelerate animals' growth and protect ecotypic enviroment. So it had great practical significence and potential application in feed,foods,medicine industry and enviroment conservation.Study intentions: It could not only increase the production of phytase in wide range with the techonology of genetic engineering, but also rebuild the characteristics of protein to obtain improved phytase according to the demand .At present, only developed Europe and America countries had the commercial recombinant phytase.Howerver they were expressed in Pichia pastoris almost .It had the behaviors :cost price of produce was so high, the cycle of produce was so long ,the technique was so complicated.So the spreading and application of phytase was limited.In our country,the application and background investigation for genetic engineering of phyA gene was no more. Some scientists study on phytase gene by adopting fixed pointmutation ,randomly mutation and constructing a new type expression system .But the effection of expressional phytase for bearing high temperature and standing the acid was not significant.Now no really production of recombinant phytase was sale in the market.Phytase was a biology comprehensive nutrien Factor .In practise and application ,people very wished the phytase could posses enzymology propert for industrialized manufacturing.With the developme nt of phytase genetic engineering,the expression of recombinant phytase protein was a new hot spot by different expressional vectors .This research selected the strong functional Prokaryotic expression vector pET30a+ and pMG36e.So using the expression vector , successfully constucted the gene engineering strains Ecoli.BL21-pET30a+-phyA and lactobacillus MG1363-pMG36e-phyA. Then we wished to obtain the phytase of mass high activity ,obearing acids and good stability .And provided basic datas and science accordings for developing a new type of multi-functional ecological agent ,which had much phytase. Up to now,there had no reports about using the two expression vector for expressing phytase. Methods,Contenst,Experiment results:1.To obtain the primitive stains with high activity, we utilized the ultra violet ray(UV) for inducing the Aspergillus niger strains ,which was separated and identified by ourselves.Result: we got the Aspergillus niger F246 with the activity of phytase about 135.483U/mL .2.Using the primer5.0 soft to design two parts of primers,directly PCR amplified the genome DNA of the Aspergillus niger F246.The production was the maturation peptide of the phyA gene.After sequncing the phyA gene,it was confirmed that the lengh of phytase gene was 1347bp.The sequnce was a new phytase gene.Its amino-acids sequence showed that this gene encoded a peptides of 467 amino-acids. Sequence analysis deduced that there were 5 potentional N-glycosylation sites within phyA gene.3. In this study we constructed two expression systems: E.coli.,lactobacillus. phyA gene was isolated from pMD18T- phyA with restriction enzyme and directly subcloned into expression vector pET30a+ and pMG36e.And constructed two new type of expression vector pET30a+-phyA and pMG36e-phyA.The two new recombinant vector pET-30a+-phyA and pMG36e-phyA were transformed into the E.coli strain BL21(DE3) and lactobacillus MG1363 by electroporation method . Then we obtained the genetic engineering strains:BL21-pET30a+-phyA and MG1363-pMG36e-phyA.4. Using the E.coli. /phyA expression system, could express the phytase for nvestigation. Moreover optimizing the expression condition ,and getting the best expression condition : the pattern of expression was inter-cell expression,the best inducing time was 3h, the best inducing concentra...