Technology Of Auricularia Gel Carrier To Immobilize Alkaline Protease

Auricularia is very rich in China,and has many functions of physiological activity.It is focused on the extraction and functional studies of Auricularia at home and abroad,there is little research of other parts.At the same time,the new carrier has gradually developed in researches of immobilized enzyme.The purpose of the research has been the development and utilization of preparation about the new immobilized carrier with Auricularia gel and made it as the carrier of the immobilized alkaline protease,then established a fixed model.The main results of this paper are as follows:(A) Preparation of Auricularia gel and screening experiment of the carrier.Extracted Auricularia respectively with alkali,acid and organic solvents in different conditions,selected the extraction condition,there respectively have got the non-alkali-soluble, non-acid-soluble and non-ester-soluble Auricularia gel carrier.And in turn to use three types of solvent extract the powder of Auricularia to get the slightly soluble Auricularia gel.According to single factor test with the extraction ratio to select the extraction conditions are:extract 30min with 3%NaOH solution at 80℃;extract 40min with 6%HCl solution at 70℃; ultrasonic extract 30min with 40%acetone solution.In terms of the immobilized enzyme activity,stability,and pH value of enzyme activity,AG-4 is more suitable for alkaline protease of immobilization from considering the stability of immobilized alkaline protease.(B) Reseach of AG-4 immobilizing alkaline protease in cross-linking method.Measure enzyme activity preservation rate to choose immobilization conditions of AG-4 fixing alkaline protease.With screening of single-factor test the response surface method(RSM) has got the best process conditions of the immobilized alkaline protease:the glutaraldehyde concentration is 12.9%,the cross-linking time is 2.5h,pH value is 9.4,and the added volume of enzyme is 4.1mL.The rate of preservation activity which have the tests been adopted is 50.89%.It is 99.47%of the predictive value.We have established a good cross-linking building of immobilised alkaline protease.(C) Immobilize alkaline protease with M-AG-4(Methacrylic acid modified AG-4) as the carrier.By initiator concentration,the concentration of methacrylic acid,the reaction temperature and reaction time as factors,we select the process conditions which are used to modify AG-4 from Methacrylic acid with the grafting rate as the target.By the screening of the single factor test,there design L₉(4³) orthogonal test to select the better conditions on the modified AG-4: initiator concentration is 12mol/L,concentration of methyl is 3mol/L,the reaction temperature is 50℃,and reaction time is 3h.The grafting rate can be reach 94.63%. M-AG-4 as a carrier has immobilized the alkaline protease.With taking the added volume of enzyme,immobilization time and pH value as the factors,the response surface analysis on the conditions which have selected by the single factor test has got the best conditions of immobilized the alkaline protease:the added volume of enzyme is 4.239mL,immobilization time is of 2.987h,and pH value is 9.133.Tests have been adopted to preserve the activity rate of 45.89%to get the predictive value of 98.08%,we have established a good covalent binding building of immobilized alkaline protease.(D) To analysis about characteristics of AG-4 and M-AG-4 immobilizing alkaline proteaseAnalysised the characteristics about immobilized alkaline protease with AG-4 and M-AG-4 as the carrier,we can find:(1) Reaction temperature of the two immobilized enzyme is higher to compare with that of the free enzyme;(2)The stability of the alkaline protease which has immobilized is better,the stability of AG-4 as carrier to fix is better than M-AG-4;(3) The optimum pH value of the two immobilized enzyme is higher than the free enzyme;(4) The number of cycle batch reaction to use AG-4 to fix alkaline protease can be as high as 7 times, and the number of cycle batch reaction to use M-AG-4 to fix alkaline protease can be as high as 5 times.We can have the conclusion by test:the enzyme preservation activity rate of the immobilized alkaline protease by AG-4 is higher than that by M-AG-4.At the same time,the effect of AG-4 fixing alkaline protease is better than that of M-AG-4 by analysises of the immobilized enzyme characteristics.Therefore it select the cross-linking method by AG-4 as a carrier for immobilization of the alkaline protease would be better.