S8 Screening Of Aspergillus Niger Strains Produced Phytase And The Phytase Gene Amplification And Identification

This paper has mainly reported that 19 strains Aspergillus nigers have been selected from 37 samples in PDA medium, among them 17 strains produced phytase in acid condition(PH5.5),7 strains produced phytase under neutral conditon(PH7.0). The best high activity and stability was S8 coming from mangrove after fermentation .Its activity of phytase in acid condition was 19.90U/ml. Used hydroxybenzene/chloroform abstracting,lysozyme breaking its cell wall and Vitagine Kit changed to abstract genome DNA from S8 Aspergillus niger mycelium, the best way was modified Vitagine Kit. Two pairs of primers have been selected by using the software of designing,meantime ,using one pare of primers reported as comparision, the best result of PCR was the primer got rid of the PhyA intron and brought KPn I and Xbal I enzyme digestion site in its 5'- and 3'-.The length of the amplified fragment was about 1500bp. After choosing the PCR system and program,the best condition was 10 buffer2.0uh MgCl2 2.5mmol/L, template (5ng/ul) 1.5ul, each primer 1.25umol/L, 4dNTP mixture(10mM/each) l.5ul, Taq DNA polymerase (5U/ul)0.5ul, denatured H2O 10.5ul, the total volume was 20ul. the best way of Puring and recovering the amplified fragment was PCR Fragment Recovery Kit after using three ways. The enzyme digestion of the PCR product proved the amplified fragment was phyA gene.