The Breeding Of A Phytase High Producing Strain, And Studies On The Purification And Characterization Of Phytase

It was that the thesis started with six strains of Aspergillus niger and one strain of Aspergillus ooryzae( preserved in the laboratory )together with more than thirty soil samples collected. We obtained one strain of Aspergillus niger( named QY )that could produce phytase by way of clarity-circle method after prescreening and re-screening. The optimized conditions for the preparation of Aspergillus nigerQY protoplasts were achieved by optimizing conditions: myceliums were cultivated in liquid medium for nine hours; and then they were disassembled in a mixed enzymes with the proportion of helicase and cellulase was 7:3; the temperature was 37℃and the procedure lasted for 2.5 h; The pH of the solution was 6; 0.7 mo1/L NaCl was used as stabilizer to balance the osmotic pressure. By mutagenesis using ultraviolet radiation of protoplasts and chemical mutagen, a more productive strain was obtained: Aspergillus nigerQY-17, cultivated under 30℃, 220 r/min for 3 d, the enzyme activity was 389.4 U/mL, 5.17 times of the initial strain.By optimizing the culture medium and fermentation conditions, the most productive culture medium for Aspergillus nigerQY-17 was to use glucose as carbon source with the recruitment of 3 g/100 mL; corn steep liquor and NH4NO3 were used as nitrogen source with the recruitment of 1.5+0.25 g/100 mL; the optimal recruitment of MgSO4·7H2O was 0.07 g/100 mL. The most productive conditions were 31℃, 220 r/min, triangular flasks( 250 mL ) with a content of 40 mL, the inoculating volume was 10%, initial pH of the medium was 6, be incubated for 72 h, Aspergillus nigerQY-17 cultivated under the optimized conditions could reach a enzyme activity of 579.8 U/mL, which was 1.49 times of the initial condition and 7.7 times of the initial strain in terms of enzyme activity.The characteristics of liquid enzyme acquired by centrifugal treatment( 7000 r/min, 20 min )were investigated when considering the application of phytase, and found that two maximal activities of enzymes appeared when the pH 2.5 and 5.5 at the temperature 37℃. Phytase manifested a good stability( >70% )when the pH altered within the range of 2 to 8. It would be deactivated promptly when the pH>8. This implicated that the enzyme could counteract acidity and neutral condition while sensitive to alkalescence. The optimal temperature of phytase was 55℃, and possessed a good enzymatic activity from 37℃to 60℃, deactivated when the temperature exceed 60℃. Thermal endurance was preferable, activity remained at 80% approximately after kept in 60℃ for 10 min. The enzyme showed little sensitivity against pepsin. When disposed with pepsin( 1 mg/mL, 37℃, pH 2.5 )for 2 h, there was 87.9% of activity left. That's all important in terms of keeping enzymatic activity in alimentary canals. Phytase could be restrained by K+, Na+, Ca²⁺, Mg²⁺, Fe²⁺ at high concentration and couldn't be restrained by Zn²⁺, Cu²⁺, Mn²⁺ at any concentration. The Km of phytase against sodium phytate was 0.5 mmol/L( pH 2.5, 37℃), Vmax was 500 nmol/mL·min; The Km of phytase against sodium phytate was 0.6 mmol/L( pH 2.5, 55℃), Vmax was 1000 nmol/mL·min. It was found that the larger affinity to sodium phytate at pH 2.5 and 37℃.Phytase produced by Aspergillus nigerQY-17 was purified:fermentation broth was treated after centrifugalization( 7000 r/min, 20 min ), acetone precipitation, DEAE FF ion exchange column and SuperdexTM75 gel column, and then two isozymes( enzymeâ… and enzymeâ…¡), single band was showed in SDS-PAGE, their molecular weights were 41 kD and 68 kD separately. The total recovery was 4.16%.