Isolation And Purification Of Neutral Protease From Aspergillus Oryzae Y1 And The Effect Of Cu²⁺ On Its Characterization

As the earliest protease used in industrial production,neutral protease is widely used in food,medicine and biological industry.In this experiment,the Aspergillus oryzae Y1 with high neutral protease was isolated from naturally fermented broad bean.The neutral protease of electrophoretic purity was purified by ammonium sulphate precipitation and DEAE-Sepharose Fast Flow chromatography.It is found that most metal ions can enhance the activity of neutral protease,and Cu²⁺activation is the most effective.The best concentration of Cu²⁺is screened out by single factor experiment and the effect of Cu²⁺on the characterization of neutral protease was investigated.In order to apply it to production,the effects of Cu²⁺on the growth and fermentation characteristics of Aspergillus oryzae Y1were investigated.The main results are as follows:?1?The naturally fermented broad bean was used as the sample,there were 6 strains from the 18 strains isolated produced hydrolytic circle on the neutral casein medium.The two strains with larger hydrolytic circle were screened,and the strain Y1 was obtained by comparing the ratio of the diameter of the hydrolyzate to the diameter of the colony,which was 2.54,and the activity of neutral protease was the highest,which was 342.42U/mL.The strain was identified as Aspergillus oryzae by morphology characteristics and 18S rDNA sequence analysis.?2?The neutral protease from Aspergillus oryzae Y1 was purified by ammonium sulphate precipitation and DEAE-Sepharose fast flow chromatography,which resulted in a10.0-fold increase in the specific activity?2264.3 U/mg?and a 21%of recovery.The estimated molecular mass of the purified protease was about 45 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.?3?The optimum reaction pH and temperature of purified protease was 7.0 and 55?,respectively.And the enzyme activity was stable at pH7.0⁷.5 and 55?,respectively.The four protease inhibitors have different inhibitory effects on the neutral protease from Aspergillus oryzae Y1.The study of the effects of metal ions on the activity of protease showed that Na⁺,K⁺,Fe²⁺,Ca²⁺,Sn²⁺,Pb²⁺,Al³⁺and Mn²⁺can activate the protease,and the activation effect of Cu²⁺was the most significant,and its relative the activity increased by 225.99%;Zn²⁺,Mn²⁺and Ba²⁺showed inhibition.The Michaelis constant of neutral protease from Aspergillus oryzae Y1 was 20.0769mg/mL,the maximum reaction rate was256.4103 g/mL·min.?4?The optimum reaction pH and temperature of neutral protease from Aspergillus oryzae Y1 were not changed adding Cu²⁺,but the activity and stability were improved.The inhibition of the four inhibitors was all reduced,and the effect of metal ions was basically consistent with that of the blank group.Under the same conditions,its Km and Vm were14.0009mg/mL and 649.3506g/mL adding 0.2mM Cu²⁺,respectively.?5?The number of spores of Cu²⁺group and control group reached the maximum of 8.77x 10⁸CFU/g and 6.63 x 10⁸CFU/g at 48h,respectively.The enzyme activity of Cu²⁺group and control group reached the maximum of 519.93U/mL and 402.56U/mL,respectively.The sensory evaluation results showed that:the Cu²⁺group was higher than or equal to the control group.In conclusion,the strain Y1 with high production of neutral protease was isolated from naturally fermented broad bean by using casein medium and the strain was identified as Aspergillus oryzae by morphology characteristics and 18S rDNA sequence analysis.The neutral protease of electrophoretic purity was purified by two purification methods.The study found that the thermostability,acid and alkali resistant and casein substrate affinity of neutral protease was improved by 0.2 mM Cu²⁺.It can also promote the germination and increase the activity of neutral protease from Aspergillus oryzae Y1.