Breeding Of High-yield Phytase Strain And Culture Conditions

An Aspergillus strain with high yield of phytase was isolated and selected from more than 200 natural samples , the yield of phytase is 5.868 IU/mL,which was identified and named Aspergillus niger Px.Secondly, the formation and regeneration of the protoplast from Asperigillus niger Px with high yield of phytase was studied. The highest yield of protoplast and the highest regeneration ratio were 9.7x105/ml and 28.2%, respectively, when the mycelia growing on the condition and liquid culture for 18 hours were dealt with 0.3% P-mercaptoethanola and compound enzyme together by using 0.7mol/L NaCl as the osmotic pressure stabilizer for 2.5 hours at 34C.The compound enzyme was composed of snailase and celluase at the best concentration ratio of 7:3, pH value 5.0.The proprieties of enzyme solution were studied at the same time. The optimal temperature and optional pH were 45 C and 5,respectively. When the enzyme is disposed at temperature 55C or pH 3.0-7.0,the relative enzyme activity is above 80%.The enzyme activity is greatly inhibited by Zn2+ AL3+ Mn2+ Cu2+ ,but Mg2+ has activation effect on phytase activity at certain concentration.The protoplasts and spores of the origin strain Px was treated by laser and UV radiation to obtain mutant strain with high phytase activity. Strain Py was obtained and enzyme activity of phytase was 9.274 IU/mL, which was about 1.58 times that of the original strain.Then the media and fermentation condition were studied through placket-burman design and Response Surface Analysis .The result shows that the optimal media were as follows:peptone 0.2%, glucose 2.57%,Sulfuric acetate 0.299%, magnesium sulfate 0.05% , manganese sulfate 0.003%, Ferrous sulphate 0.003% , potassiumi chloride 0.05%, phytic sodium 0.236%.On this condition, theoptimum culture condition of Aspergillus strain Py producing enzyme were as follows: temperature 31.1 C, fermentation period 4.7d,pH 5.6. After the above optimum, the yield of phytase is 15.73IU/mL.At last, the phytase was used to hydrolyze sodium phytate in the experiment ,the rate of removal is high up to 88% at 4 hours.