Screening Of Alkaline Protease Strain And Characterization Of Enzymatic Properties

Alkaline protease refers to a protease that has peptide bond hydrolysis activity in the neutral to alkaline pH range.With the improvement of people’s awareness of environmental protection,the use of environmentally friendly processes is advocated in industrial production,which provides a good opportunity for the promotion and use of alkaline protease in industrial biotechnology.Alkaline proteases widely exist in animals,plants and microorganisms.Due to the limited resources of animals and plants,screening of microorganisms that can produce alkaline proteases has become a hotspot in the development of biological enzymes.In this study,a silt sample was taken from the Hainan offshore shellfish breeding base,and a strain F3 that produced alkaline protease was isolated and screened.Taking this as the research object,the strain identification and fermentation enzyme production conditions were optimized;The alkaline protease was identified by mass spectrometry and the enzymatic properties were studied;the removal effect of this enzyme in detergents on protein stains was explored,and the theoretical support for the application of alkaline protease in the washing industry was provided.The results of the study are as follows:（1）The casein plate method was used for primary screening and the Folin-phenol method was used to measure the enzyme activity.After morphological identification and molecular biological identification,it was determined to be Serratia marcescens.Through cytotoxicity experiments,leaf infection experiments and acute toxicity experiments in mice,it was proved that the strain F3 was not toxic to macrophages,plant（cassava,rice,peanut,yam）leaves and mice.（2）Through single factor test and Plackett-Burman test,it was determined that pH,galactose and Na Cl had significant effects on enzyme production.The center point of the response surface experimental design was determined by the steepest climbing experiment,and finally the optimal fermentation enzyme production conditions of strain F3 were determined by response surface analysis:galactose 11.09 g/L,initial pH 8.4,peptone 20 g/L,Na Cl 5.04 g/L,inoculum volume 0.01 OD₆₀₀,K₂HPO₄ 10 g/L,liquid volume 150 m L/500m L,temperature 30°C,and incubation time 48 h.After optimization,the alkaline protease activity of strain F3 was 339.36±4.3 U/m L,which was 2.21 times higher than that before optimization.（3）Through the four-step separation and purification process of fermentation broth centrifugation,50%saturation ammonium sulfate precipitation,dialysis,and anion exchange chromatography,an alkaline protease with a molecular weight of about 50 KDa was successfully obtained.Its enzymatic properties were explored.The optimal catalytic temperature was 40～50°C,the optimal pH was 9,and it had good thermal stability and alkali resistance;5 m M and 10 m M Mg²⁺,Ca²⁺,Co²⁺significantly promoted the enzymatic activity,while Fe²⁺and Li⁺have obvious inhibitory effects on the enzyme activity;1%（v/v）H₂O₂and SDS have no significant effect on the enzyme activity.The protease is intolerant to Tween-80 and Triton-X100,and Tween-20 is not resistant to the enzyme.It has a promoting effect;the enzyme is intolerant to most organic solvents,and only 20%and 30%（v/v）n-hexane has a certain promoting effect on the enzyme activity.（4）Through the washing performance test,it is proved that adding the enzyme to the Tide detergent solution can effectively improve the removal effect of blood stains and egg yolk stains on cotton fabrics.It was proved by SEM,EDS and FTIR that the surface morphology,chemical element composition and structure of cotton fabrics were not affected before and after enzyme treatment.