Study Of Fermented Conditions Of Producting Phytase By Aspergillus Ficuum In Vinegar Residues And Phytase Purification

Vinegar residue was the by-prodct in vinegar processing and it was discarded for its high content of fiber. That behavior results in the wasting resource and environmental contaminated. But vinegar residue is the secondary resource. Producting phytase by Aspergillus ficuum(CGMCC 3.4322) in vinegar residue was doable in our former studies. For screening and gaining the higher activities and qualities of phytase in vinegar residue, we isolated and purified the phytase of mutant, optimized the fermented conditions of mutant basing on the original A. ficuum.Exp. One Aspergillus ficuum with producing phytase mutantion breedingAspergillus ficuum was disposed with ultraviolet and nitrosogudnidine for the mutant of higher activities and quantities.We choiced the mutant that its value of ratio of transparent circle and colony diameter(HC value)was bigger than original strain by phytase screening medium. We gained 49 mutants of A. ficuum after radiating for 10-100s verdically with ultraviolet lamp(30 W). We gained 50 mutants of A. ficuum after shaking for 3-30 min under 0.5 mg/mL NTG at 30℃. A. ficuum NTG-23 that its activities were 274% of original strain in secondary screening was confirmed to the subject for futher studies.Exp.Two Study of isolating and purifying phytase from A. ficuum NTG-23 and its propertiesA phytase with a molecular mass of 65.5 KDa, was isolated from mycelium of the mutant A. ficuum (NTG-23) with a procedure which involved anion exchange chromatography on DEAE-Cellulose, cation chromatography on CM-Cellulose and gel filtration by fast protein liquid chromatography on Superdex 75 16/60. Enzymic properties of isolating phytase from mutant of A. ficuum: optimum pH was in the range between 1.3 and 1.4; optimum temperature was in the range between 65℃and 70℃. Phytase was slightly sensitive to Ca²⁺,Fe³⁺,Al³⁺,Mn²⁺,Mg²⁺,K+,Zn²⁺,Cd²⁺,Cu²⁺,Pb²⁺ and Hg²⁺; EDTA enhanced the phytase activity at the concentration of 1.25-10.0 mM for about 10%. Upon incubation of the purified phytase with various sodium phytate concentrations (up to 5.0 mM)the reactions were found to follow Michaelis-Menten kinetics, displaying Km and Vmax values of 0.295 mM and 55.9 nmol/min, respectively. The phytase showed strong resistance toward pepsin and trysin. It remained more than 90% of total phytase activity after incubated in the protease solution for 60 min. The isolated enzyme showed strong temperature stability at 60℃. There was only a slight decrement in activity when the temperature was increased from 70℃to 80℃.The isolated enzyme showed strong temperature stability at 60℃. The phytases showed a steady loss of activity with increasing time at 70°C for more than 10 min, 90% activity reservation for 60 min. The phytase activity vanished after incubated at 80°C for 10 min.The N-terminal sequence of the phytase by EDMAN degradation was as followed: F S Y G A A I P Q S T Q E K Q. The identities of the N-terminal amino acid sequence of the phytase of A. ficuum NTG-23 was 100% with amino acid sequence of A. niger NRRL 3135 PhyB. We confirmed it was acid phosphatase preliminary.We found 9 acid phosphatase phyB sequences that their identities were 85% after cloning phyB of A. ficuum NTG-23,sequencing and blasting on line. The phyB sequence of NTG-23 and DQ297678 were 90% with phyB sequence of A. ficuum NTG-23.The sequences of DQ297678 and DQ787156 were alignmented. Results showed: phyB of A. ficuum NTG-23 was located in from the first to the third codon sequence; there were 3 bases different in the first codon sequence with DQ297678 but there were 2 bases different in the first codon sequence with DQ787156; there was no different base in the second codon sequence; there was 1 base different in the third codon sequence with DQ297678 and DQ787156.Exp.Three Study of Fermented Conditions of Producting phytase by A. ficuum NTG-23 in Vinegar ResidueWe optimized the fermentd conditions of vinegar residue for higher phytase activities and quantities. After screening the 11 factors which effect phytase activities and quantities, we found three factors that its'reliability was over 90%: glucose, soybean meal, fermentation period. We actualized the steepest ascent according to step size that it was dued to the regress analysis of the Plackeet-Burman results. Results showed that the response value (activities of phytase)approached the maximum value area when the value of glucose,soybean meal and fermentation period were 1.5%, 0.7% and 96h respectively. Responce surface method was statistic in the central composite design. We found the fitting regession equation as followed: Y=67.76+4.09X₁+19.14X₂+18.53X₃-6.03X₁²-10.88X₂²-9.93X₃²+3.38X₁X₂+1.06X₁X₃+7.09X₂X₃The R2=95.99% of the fitting regression equation, lack of fit test was not significant. That showed that the fitting regression equation was crediable. We found the maxmum anticipant response value was 98.19 U/g DMR when the value of glucose, soybean meal and fermentation period were 7.2%, 5.1%, and 271h through the solving the first derivative equation. The activities of phytase were 98.37 U/g DMR under validating experiments. We confirm that the analysis was crediable with fitting regression equation when true experiment was replaced. The optimum fermented conditions'combination of producting phytase by A. ficuum NTG-23 in vinegar residue was the every variable value on maximum value of fitting regession equation.