Study On Screening Of Thermostabilable Alkaline Protease Strain,Optimization Of Fermentaion Condition And Its Enzymatic Properties

Alkaline protease is a group of important proteolytic enzyme which hydrolyze protease into amino acids and small peptides,and widely used in many industries such as food,medicine,washing and leather.Keeping in view sustainable development,much work is focused on using ecofriendly enzymes in place of hazardous chemicals in industrial processes.It is the need to find the high yield strain of alkaline protease which can be used in industrial production,reduce production cost and establish the production system of alkaline protease.Therefore,in order to promote the quality of the alkaline protease and expand the application of alkaline protease in China,the screening of high yield alkaline protease strain,and its enzymatic properties,still is the indispensable way to realize the industrialization of production and application.The paper is based on the soil of peach trees,meadows,jungles and landfills,screening the strain ZJ1502 of high producing alkaline protease and then identification of strain,the optimization of fermentation conditions and the separation and purification of alkaline protease and its enzymatic properties were researched.it aims to provide a theoretical basis for its practical application.1.In this study,strains were isolated from different areas in selective skim milk medium,and then with the Folin-phenol detection,twenty of them which had clear zone on casein agar plate were fermented in flasks.One strain named ZJ1502 producing high yield of alkaline protease was screened.Based on morphological observation,physiobiochemical characteristics reaction and phylogenetic analysis of 16S rRNA of the strain,it was identified that the strain ZJ1502 was belonged to Bacillus weihenstephanensis.2.To improve the enzyme activity of strain ZJ1502,the effects of the medium components and the fermentation conditions for the alkaline protease production in Bacillus sp.ZJ1502 were studied.Nine factors were firstly evaluated by the Plackett-Burman design on the basis of single factor experiments,and yeast,glucose and pH were found to play a very important role in the production of the alkaline protease.Their optimal levels were further determined by the Box-Behnken design and response surface methodology.The optimal medium components for Bacillus sp.ZJ1502 to produce alkaline protease were as follows:yeast 6%,glucose 5.61%,K2HPO40.9%,KH2PO4 0.2%,MgSO40.4%;and the optimal fermentation parameters were pH 8.94,inoculation 6%,temperature 37?,and liquid volume 50 mL/250 mL.The activity of alkaline protease was up to 153.04 U/mL under the above conditions,a 3.77-fold increase than that in the original medium.3.Under the optimum fermentation culture conditions,an alkaline protease from Bacillus sp.ZJ1502 was purified by using a three-step separation procedure based on ammonium sulfate precipitation,DEAE-52 cellulose ion exchange chromatography and Sephadex G-100 size-exclusion chromatography with 18.57-fold increase in specific activity and 7.13%increase in the recovery.The molecular mass of the alkaline protease was estimated to be 14.3 kDa by SDS-PAGE analysis.4.The optimal pH 10 and temperature of 40? obtained for the purified protease,The protease showed a strong stability in a wide range of pH values ranging from 9-11.The metal ions have different effects on the activity of the purified alkaline protease,It was significant that the addition of Mn2+ could increase its activity.Sodium dodecyl sulfate(SDS)and ethylene diamine tetraacetic acid(EDTA)were found to inhibit the activity of the alkaline protease,The activity dramatically decreased with their concentration increased.Tween-60 and Triton x-100 had almost no effect on the activity of alkaline protease,while Twin-80 could obviously promote the activity of alkaline protease;non-ionic surfactants had less effect and even enhanced on the activity when compared to ion surfactant.Additionally,the purified alkaline protease exhibited poor tolerance in organic solvents such as N-butanol and ethanol,the activity was remarkably improved in glycerol and oleic acid.the enzyme showed a certain resistance in 0.5 mol/L H2O2.Casein was the most suitable substrate for the alkaline protease,which can hydrolyze some bovine serum albumin and cannot hydrolyze gelatin and collagen.The Km and Vmax values of this enzyme were 16.7 mg/mL and 14.71 ?g/(min mL),respectively.With a potential thermal stability and alkali resistance of an alkaline protease can provide theoretical basis for the future in related fields.