Screening Of Protease-producing Strains And Study On Their Enzymatic Properties

Proteases have a wide range of applications in industries such as food,medicine,washing,and tanning.In this thesis,a protease-producing wild strain CL-10 was isolated and screened from the canteen sewage of Nanchang University,and the strain identification,fermentation process optimization,mutagenesis,separation and purification,and enzymatic properties were studied.The results are as follows:From the sewage samples of the canteen of Nanchang University,the protease enzyme activity was determined by strain enrichment,plate primary screening,and shake flask rescreening to obtain a strain with higher protease activity,named CL-10.It was named Bacillus amyloliquefaciens CL-10 by morphological observation,scanning electron microscopy,Gram staining,physiological and biochemical tests,and molecular biology tests.Through single factor experiment and response surface optimization of the medium composition,the best fermentation medium conditions were explored:rapeseed meal content 6.1%,corn flour content 4.4%,bran content 3.2%,ZnSO4·7H2O concentration 0.2%,vomiting The Tween 20 concentration is 0.7%;the optimized fermentation conditions are:culture temperature 37?,initial pH 7.0,filling volume 50 mL,inoculation volume 4%,fermentation time 54 h.Compared with before optimization,under optimal conditions,The enzyme activity increased from 2715 U/mL to 6648.4 U/mL.Carry out UV-diethyl sulfate compound mutagenesis.The optimal mutagenesis condition of ultraviolet light is power 15 W,irradiation distance 30 cm,irradiation time is 150 s;the optimal mutagenesis condition of diethyl sulfate is 0.5%concentration,temperature 37?,rotation speed 200 rpm,mutagenesis time 14 min.After UV and diethyl sulfate compound mutagenesis,a strain Bacillus amyloliquefaciens CL-10-1 with a protease production of 7581.3 U/mL was finally screened,which was 14.03%higher than the starting strain.The strain was cultured for 5 consecutive generations.The protease activity of its fermentation broth retains more than 97%compared with the first-generation strain,and has good genetic stability.The pure enzyme detected by electrophoresis as a single band was separated and purified by ammonium sulfate precipitation,dialysis,DEAE-Sepharose Fast Flow,and Sephadex G-50.The molecular weight of the protease was 27.9 kDa compared with the standard protein through software analysis.To study the enzymatic properties of the protease produced by Bacillus amyloliquefaciens CL-10,the optimum temperature of protease is 45?,and the stability is good at 35-45?.It can retain more than 50%of the enzyme activity after 90 minutes of heat preservation;The optimal pH of the enzyme is 7.0,and the stability is better at pH 6-8.It can still retain more than 50%of the enzyme activity after 120 minutes of incubation.