Study On Preparation,Properties And Its Application Of Pineapple Peel Protease

ObjectiveIn this paper,use waste pineapple production of pineapple peel as raw material,extraction,separation and purification of the bromelain,and stability of the pineapple protease from the three aspects of storage conditions and physical and chemical factors were analyzed by measuring the Michaelis constant,half-life and activation energy of heat stabilizing effect of the three parameters of pineapple peel protease and protease,pineapple peel applied in enzymatic hydrolysis of fish protein,enzymatic hydrolysis conditions were optimized.At the same time,the Michaelis constant solution of pineapple peel fish scales protein enzymatic analysis,not only saving resources,increase economic benefits and environmental benefits,but also for the development of new sources of protease and its application in the actual provides scientific basis and experimental foundation.MethodsUsing single factor experiment method,selection of liquid material ratio(v:m),p H extraction and extraction temperature(?)three main factors as the object of study,the content of protease(mg/g)as the evaluation index for response surface analysis,optimization of process parameters of pineapple protease extraction;by ammonium sulfate precipitation,dialysis desalting and carboxymethyl cellulose(Carboxymethyl Cellulose,CMC)column chromatography on pineapple protease was isolated and purified to obtain the purity of enzyme and enzyme activity is relatively high,is the purification of the purified protease enzyme;UV spectrum scanning.The degree of Purification Identification of the enzyme,then through HPLC qualitative and quantitative analysis further the purified enzyme;extraction,separation and purification of the final calculation of each stage of the protease content,enzyme activity,specific activity,purification and enzyme recovery.With purified enzyme as the research object,In order to study the effects of different storage conditions,physical and chemical factors on the enzymatic activities of pineapple peel by single factor experimental method,the stability conditions of the protease were obtained.Of pineapple peel purified enzyme thermal inactivation properties by drawing the double reciprocal curves were obtained with casein Michaelis constants for the substrates,by drawing heat inactivation rate curves obtained for pineapple protease reaction rate constant K,by different K value is calculated under the different temperature(T),half-life T(1/2),and then to LNK of T-1 mapping,according to Arrhenius equation of enzyme reaction activation energy delta e.Preliminary application of pineapple protease enzyme solution fish scales protein and the degree of hydrolysis(DH)as the evaluation index,On the basis of single factor experiment,the response method was used to optimize the concentration of substrate [S](%),enzyme solution temperature(DEG C),the enzyme solution time(min)and enzyme dosage [E/S] four main factors of enzymatic hydrolysis of fish protein effects,to solution of the optimal parameters of the fish scales protein enzyme and of pineapple peel protease enzyme solution the Michaelis constants for fish scales protein do preliminary analysis.Results1,The use of response surface methodology pineapple peel crude protease extract the optimal process parameters optimization,material to liquid ratio of 3:1,extraction temperature of 50 DEG C,extraction p H 6.0 and extraction time 50 min.Through the optimum conditions obtained pineapple peel of the crude protease content for 0.937±0.012mg/g,respectively,the enzyme activity was 1.823U/ m L.2,Using ammonium sulfate salting out method purified pineapple protease to obtain optimum salting out percentage is 30%;after dialysis and CMC chromatography purification pineapple protease purity reached 90.08%,the concentration of 223.62ug/ml.3.The best storage condition of pineapple skin protease was the best stability under the condition of low temperature storage,water content less than 20%,and storage time less than 4H.4.The results of the study on the stability of pineapple peel protease showed that the enzyme had better stability when the temperature was less than 25 ? and p H was between 6 and 3;5,chemical factors on stability of the pineapple skin protease research results show that Ca2+,EDTA,glucose,xylose,maltose,tartaric acid potassium sodium,sodium citrate,nitrite sodium and citric acid on pineapple protease have strong activation effect;thio sulfate sodium,Na+,K+,sucrose and starch had no significant effect on pineapple protease activity;Fe3+,Al3+,methanol,ethanol and n-butanol on pineapple protease has significant inhibitory effect.6,pineapple protease thermokinetic research results show that,the pineapple protease Michaelis constant(km)for 5.122g/L,45,55,65 and 75 DEG C four storage conditions,pineapple protease with a half-life t(1/2)were gradually decreased,by the Arrhenius equation for the reaction activation energy delta e is 2.6 x 103J/mol.7,through single factor experiment and response surface analysis method to obtain pineapple protease enzyme solution fish scales protein of the best conditions of enzymatic hydrolysis of substrate concentration [S]5%,enzyme solution temperature of 70 DEG C,and enzyme solution time 2.5h,plus the amount of enzyme [E/S]9%.Under the optimum conditions of hydrolysis degree as 1.314±0.040%;pineapple protease enzyme solution the Michaelis constants for fish scales protein was 4.11%.ConclusionsThe results show that the higher content of pineapple protease,has high activity,enzyme content under the optimal conditions was 0.937±0.012mg/g,the activity of 1.823U/m L;after separation and purification of the enzyme activity was significantly increased by identification of better purification,the purity was 90.08%,purification fold was 10.26;stability results show that at low temperature to avoid light storage,the moisture content is less than 20%,the storage time is less than 4h,p H has better stability in 3~6 between the conditions,Ca2+,EDTA,D-xylose,glucose,maltose,potassium sodium tartrate,sodium citrate,sodium nitrite and citric acid have strong activation effect on this enzyme;Fe3+,Al3+ methanol,ethanol and n-butanol,this enzyme had obvious inhibitory effect;kinetic studies indicated that pineapple protease Michaelis constant Km was 5.122g/L,the activation energy of E is 2.6×103J/mol.;the application of pineapple protease in fish scales protein enzyme solution has certain feasibility,degree of hydrolysis as 1.314±0.040%,Michaelis Menten constant was 4.11%.