Study On The Modification, Immobilization Of Pectinase And Application Of Immobilized Pectinase

This topic is optional subjects. With the development of fruit and vegetablejuices and fruit wine industry, pectinase is used more widely. It has already been oneof the largest enzyme productions of world. Modifying and immobilizing pectinasecan improve the coefficient of utilization of pectinase in industry and reduce productioncosts. This is significant on improving economic efficiency. For this purpose, the mainissues in this thesis have been done as follows:1. Adopted3,5-dinitrosalicylic acid method to determine the activity of pectinase.Using one-factor-at-a-time determined the optimal absorption peak of reactiveproducts of galacturonic acid and DNS was at540nm. DNS solution of2.5mL, thecolor reactive time of4min were found optimum. The equation of galacturonic acidstandard curve was y=0.6801x. Optimal temperature and pH of pectinase were50℃and4.4, respectively. Activity of pectinase was measured to be1.894×10⁵U.2. Determined the effect of diversiform chemical reagents and metal ions on theactivity of pectinase. Studies had shown that after chemical modification of lysine anddisulfide bonds, the activity of pectinase basically unchanged. It indicated thatpectinase activity centers didn’t have lysine and disulfide. The activity of pectinasewas precipitate when tryptophan modified. This indicated that the activity center ofpectinase contained tryptophan. With EDTA treatmented on pectinase, the activitypectinase basically unchanged. This indicated that pectinase activity center didn’thave metal ions. Metal ions Fe²⁺, Cu²⁺promoted the activity of pectinase about16%and15%, respectively. Ca²⁺has slight inhibitory action on activity of pectinase.Other metal ions had no significant effect on pectinase activity.3. Collecting mycelium of Rhizopus oligosporus as raw material by fermenting toprepare chitosan, immobilized carrier of pectinase. Used soybean sprouts as rawmaterial and adopted fermentation method to collecte mycelium of Rhizopusoligosporus for preparing chitosan. Based on the one-factor-at-a-time and on principleof Box-Behnken experiment design, medium initial pH, inoculation concentration andcarbon source consumption were optimally analyzed with the Response SurfaceMethodology. The mathematical model of quadratic polynomial on optimal technicsof mycelium’s growth of Rhizopus oligosporus was developed in the ResponseSurface Methodology. The analysis of variances had showed that the optimaltechnical parameters were found to be: medium initial pH3.65, inoculationconcentration0.92%, carbon source consumption19.77g/L. The deacetylationdegree of chitosan that was prepared using deacetylated mycelium is90.87%. Yieldof chitosan is7.46%.4. Immobilized pectinase by chemical crosslinking. Using chitosan as carrier,glutaraldehyde as crosslinking agent, immobilized pectinase by chemical crosslinking method. Based on the one-factor-at-a-time and on principle of Box-Behnkenexperiment design, glutaraldehyde concentration, chitosan and glutaraldehyde reactivetime and pectinase solution pH were optimally analyzed with the Response SurfaceMethodology. The mathematical model of quadratic polynomial on optimal technicsof immobilized pectinase was developed in the Response Surface Methodology. Theanalysis of variances had showed that the optimal process parameters were found tobe: pectinase solution pH3.8, glutaraldehyde concentration0.35%, chitosan withglutaraldehyde reactive time4.22h. Prepared immobilized pectinase under theoptimal conditions and the optimal pH and temperature of immobilized pectinasewere determined to be pH3.6and55℃, respectively. The temperature stability ofimmobilized enzyme was highly improved compared to free pectinase. The half lifeperiod of immobilized pectinase was24.12d.5. Using immobilized pectinase to treatment apple pulp for improving the juiceyield. On the basis of single factor investigations, the optimal process parameters werefound to be: reactive temperature50℃, apple pulp pH3.40, apple pulp withimmobilized pectinase mass ratio1:15, reactive time3.00h. On principle ofBox-Behnken experiment design, relative temperature, apple pulp pH and relativetime were optimally analyzed with the Response Surface Methodology. Themathematical model of quadratic polynomial on optimal technics of effect ofimmobilized pectinase on apple pulp was developed in the Response SurfaceMethodology. The analysis of variances had showed that the optimal processparameters were found to be: temperature49.4℃, apple pulp pH3.43, reactive time3.50h. Under the optimized conditions, the yield of apple juice was62.421%whenimmobilized pectinase was useed on the10th. And it was about13%higher than thecontrol group.