Purification And Characterization Of Protease From Neurospora Crassa And Application Of The Fungi

Neurospora crassa is a bacterium which has a high production of cellulose and carotenoid.At present,there have been some related studies on the cellulase and carotenoid produced by Neurospora crassa,but little research has been done on the production of proteases.In this study,Neurospora crassa was used and inoculated on bean dregs for solid state fermentation.The fermentation conditions of protease production and soluble protein content,the isolation and purification of proteases,the enzyme properties and the solid state fermentation of rapeseed meal by strains were studied.The experiment results are as follows:1.The bean dregs was identified as the raw material for solid state fermentation media.The fermentation conditions of protease and soluble protein production were optimized by using the single factor method and response surface Box-Behnken combinatorial design method.The optimum fermentation conditions for producing the enzyme and soluble protein are as follows:the amount of bean dregs in the experiment was 10 g,the amount of water was 21 m L,the inoculation amount of bacterium solution was 2 mL,the culture temperature was 30°C.,the initial pH of the culture medium was 5.0 and the fermentation time was 72 h.The final protease enzyme activity was 1959.82±9.54 U/g and the soluble protein content reached a maximum?9.99±0.04 mg/g?at 72 h.The verification experiment was carried out with the best conditions obtained by the model.It was found that the obtained protease activity and soluble protein content were similar to the predicted values,indicating that the model had a good fit.2.Separation and purification of Neurospora crassa protease by using ammonium sulfate precipitation,dialysis,DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-75.The purified protease had a specific enzyme activity of 5518.37 U/mg and a purification multiple of 28.27.The recovery rate was 31.00%.A single band was obtained by using SDS-PAGE electrophoresis.It was proved that the protease was electrophoretically pure.And a single protease activity band was obtained by zymogram analysis.The purified protease was identified by LC-ESI-MS/MS to obtain the uncharacterized protein which named B19A17.360 in Neurospora crassa and shared the characteristics of common properties.The molecular weight of pure enzyme was about 30 kDa,The molecular weight of the uncharacterized protein was about 30.41 kDa and the isoelectric point was 7.83.There was one peptide segment that was matched,so it might be a new protein.The peptide fragment is RQGTNAVATAVNSLNARC.3.The enzymatic properties of the purified protease were measured.The serine protease inhibitor completely inhibited its activity.It was determined that the enzyme was a serine protease,and the best substrate was casein.The optimum temperature was 55°C.The suitable pH was 9.0,And the enzyme activity was stable at 30⁴5°C,pH 6.0¹0.0,The Michaelis constant was K_m=2.18 mg/mL,the maximum reaction rate was V_max=36.36?g.m L^-1.min^-1,The protease activity was increased by 0.5 mol/L NaCl,2 mmol/L Tween-80 and Siban,5 mmol/L Ca²⁺,1%ethanol,acetone,acetonitrile,isopropanol,10%acetonitrile and isopropanol.On the contrary,2 mmol/L SDS and Triton,10 mmol/L Ca²⁺,5 mmol/L and 10 mmol/L Cu²⁺?Co²⁺?Mg²⁺?Zn²⁺?Fe²⁺could inhibit the activity of protease.And the chemical reagents such as methanol,ethanol,acetone,and acetonitrile used in the experiment had no obvious inhibitory effect on the protease activity,which indicating that the protease had a certain tolerance to those chemical reagents.4.Neurospora crassa was inoculated into rapeseed meal for solid state fermentation for 96 h.The results showed that the contents of crude protein,contents of soluble protein and contents of total amino acid increased significantly?P<0.01?,as the extension of fermentation time,which improved by 12.50%,232.52%,724.45%,respectively.The contents of carotenoid increased from 0?g/100g to64.68±1.42?g/100g.Furthermore,the contents of acid soluble protein were increased and then down to steady,the maximum was 25.86±0.05 g/100g at 24 h.On the contrary,the contents of crude cellulose,tannin,phytic acid and thioglycoside showed significant decreasing treads?P<0.05?.The degradation rates were 23.48%,24.98%,10.20%and 14.79%,respectively.The large protein in rapeseed meal was degraded into small molecules after fermentation.It showed that the nutrition values of rapeseed meal were improved greatly by Neurospora crassa.