Study On Screening,Identification,Fermentation Process Optimization And Enzymatic Characterization Of Thermostable Neutral Protease Producing Strains

Neutral protease is one of the protease preparations first used in industrial production.Because of its mild conditions and high catalytic rate,neutral protease has been widely used in food,medicine,leather,feed,chemical and waste treatment industry.In this paper,a neutral protease producing Bacillus subtil is ZG20 was isolated from soil.The fermentation conditions of ZG20 were optimized by changing the composition and culture conditions of the medium.The crude enzyme was isolated and purified,and its enzymatic properties were studied.The main research works are as follows:(1)A strain which could produce neutral protease was isolated from soil samples collected in Dove square.It was identified as Bacillus cereus by morphological,physiological and biochemical identification and molecular biological identification,and named as Bacillus cereus ZG20.(2)The fermentation conditions of ZG20 strain were optimized.Through single factor experiment and Plackett-Burman test,it was determined that the key factors affecting the activity of neutral protease were glucose concentration,peptone concentration and pH.On this basis,the response surface test was carried out and the predicted values were obtained after verification test.The test results showed that the optimum medium group ZG20 strains are divided into:glucose 35 g/L,peptone 40.38 g/L,sodium chloride 15 g/L,Magnesium Sulfate 15 g/L,Tween-8010 g/L;the optimum culture conditions:pH7.15,inoculum 5%,medium volume 50mL/250mL,age 12h,fermentation time 36h,the enzyme activity was 698.41 U/mL.As the initial fermentation medium activity 1.77 times.(3)Isolation and purification of crude enzyme from fermentation and characterization of its related enzymes.The neutral protease precipitated by ammonium sulfate was recovered,concentrated and purified by Cellulose DE-52 anion exchange column and Sephadex-G100 gel filtration column.The purity and molecular weight of neutral protease were determined by SDS-PAGE protein electrophoresis.Isolation and purification experiments and related enzymatic properties showed that the molecular weight of neutral protease was 29kDA,the optimum pH value and temperature were 7 and 45,respectively,and 0.05mol/L had good stability in hydrogen peroxide.Mg2+,Zn2+,K+and Na+could promote the protease,but Cu2+,Fe2+and Ca2+had little effect on the enzyme.Mn2+,Al3+,Co2+and Ni2+had a great inhibitory effect on the enzyme.Tween-80 could promote the enzyme.With the increase of Tween-80 concentration,the promotion effect of the enzyme was more obvious;SDS,EDTA and TritionX-100 inhibited the enzyme in varying degrees,and the inhibitory effect of EDTA was the most obvious.The neutral protease has certain organic solvent resistance to ethanol,ethylene glycol,glycerol and other organic solvents,and has a certain degradation effect on casein,bovine serum albumin,gelatin,egg white and milk powder,and the degradation of casein is most obvious.The protease enzyme kinetics is drawn.The calculated Km is 12.74 mg/mL,Vmax is 28.57?g/(min-mL).