Studies On The Breeding Of Phytase Producing Strains Aspergillus Niger, And On The Purification And Characterization Of Phytase

Phytase showed widely useful prospect in feed, food, medicine and so on. However, the price of it was so expensive that it couldn' t be spreaded in industral. The purpose of the current investigation was to screen the strains of producing phytase , to make the conditions of producing phytase superior, to purify phytase and research the characterization of its.1. Studies on screening of strains of producing phytase and the conditions of producing phytaseThe strains of producing phytase could be identified by the hydrolysis bound in differential medium. Aspergillus niger AN010001 secreting phytase was isolated by screening and second screening. The conditions of producing phytase was studied. The results showed: The best additive water to wheat bran solid medium was 35 percent. A. niger AN01001 produced high activity phytase when it was cultivated at 30癈 from 90 to 120 hours, and obtained the highest enzyme activity at 114 hours. Phytase activity was effected by different buffer and pH, enzyme activity was high at pH 5.5 and 6. 5 when extracted by acetate buffer, but was high at pH 7. 5 used water. Ca2+ was required not only for the activity of the enzyme but for stability. The results was better when used 3 percent to extract.2. Studies on the induced mutagenesis of A. niger of producing phytaseA. niger AN01001 was induced mutagenesis by UV for obtaining the enzymatic activity as high as possible. As a result, A333strain had a 43 percent higher activity than control. However, activity raised a litter after the complex induced mutagenesin by UV and LiCl. At last, three strains of high activity was induced mutagenesis by NTG, after the routine screening procedures , activity of N28 and N118 was higher 47. 5 and 40. 9 percent than that of A. niger AN01001, and higher 29.5 and 23.7 percent than that of A199. The abilities of producing phytase were stabile after 5 transference.3. Purification and characterization of phytase from A. niger AN 01001A. niger AN01001 was inoculated on solid media and cultivated at 30℃ for 5 days. Proteins were extracted from solid-state fermentation with 50mM acetate buffer (pH5.5). The molecule weight of the phytase protein was determined as about 78KD by SDS-PAGE. The purification procedures include ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, gel electrophoresis and electroelution . To investigate the pH optimum and pH stability, the phytase assay was performed at a pH range of 2 to 8 with a variety of buffers by standard assay. The phytase had a pH optimum of pH 5. 5 and was virtually inactive above pH 6.0. When the enzyme was incubated at various pH values at 37?C for 30 min in the absence of substrate and the residual activity was measured, the phytase was found to be stable at the pH range of 2 to 8. The temperature profile of purified phytase was determined from 30 to 60℃ by standard assay at the given temperature. The optimum temperature was found to be 55℃. To investigate thermal stability, the phytase was incubated at 90℃ for 30 min in 0. 05 M acetate buffer, pH 5. 5, and its,activity was determined by standard assay, 62. 3% of the activity remained. Enzyme retained more than 67% of its original activity at thetrypsin/phytase ratio (w/w) 0.01 when the phytase was incubate at 37 for 120 min. Phytase activity can be inhibited by Cu2+, Zn2+; and unaffected by Ca2+, Mg2+, Mn2+ and Al3+.