| Enzyme catalyzed reaction have advantages of efficiency, substrate specificity, mildreaction conditions and energy saving, so it has great potential in areas such as foodindustry and bio-chemical industry. Compared with free enzyme, immobilized enzyme hascharacters such as easy separation, repeated use of enzyme and little pollution. It also canmake production continuous and reduce cost, so immobilized enzyme has been widely usedin biology, food, medicine and other fields in recent years. The catalytic efficiency ofimmobilized enzyme is closely related with the immobilization efficiency. Two importantfactors which affect the immobilization efficiency are immobilized carriers andimmobilization methods. In this paper, cationic ion adsorption carrier of immobilizedenzyme were prepared by chemical modification of cotton fibers and covalently graft withpolyethyleneimine. We studied the immobilization efficiency of the immobilized lactaseand continuous catalytic hydrolysis of lactose by immobilized enzyme.This paper developed a new ion adsorption type carrier of immobilized enzymegrafted by polyethyleneimine (PEI). The main component of the carrier is cellulose.Hydroxyl groups on the cellulose surface are oxidized to aldehyde groups by sodiumperiodate. Based on the covalent reaction between amino group of polyethyleneimine andaldehyde group of oxidized cotton fibers, the cotton fiber with surface covalently graftedwith polyethylenimine is obtaind, which can be used as supports for enzymeimmobilization.Under certain pH condition, polyethyleneimine molecules present withpositive charge, have strong adsorption of enzyme molecule with negative charge, so as toimmobilize enzymes. The oxidized cotton fiber grafted with polyethyleneimine in this testis a new type of carrier materials for enzyme immobilization. Compared with traditional ionadsorption carrier, the loading capacity of immobilized enzyme is larger in immobilizedenzyme process and adsorption has little effect on spatial structure of enzyme. Therefore,this immobilized enzyme has high catalytic efficiency.In the process of cotton fibre oxidized by sodium periodate, taking aldehyde content asthe index, we studied the effects of oxidized temperature, oxidized time and oxidantconcentration. The results showed that the best oxidized effect was obtained with30℃,10h,0.2mol/l with a aldehyde content49.43%. In preparation of PEI-oxidized cottonfibre, we researched the effects of concentration of PEI, pH and grafted time on the aminocontent on the surface of carrier. The final grafting condition was optimized and determined:6mg/ml, pH6.5,2h with a amino content on carrier surface of1.35mmol/g.We successfully used oxidized cotton grafted PEI cellulose to reversibly immobilizelactase. Factors that influenced the immobilization of lactase by PEI-oxidized cottoncellulose were also studied. The result presented the optimal condition for immobilization were as follows: support/enzyme ratio of0.3mg/ml,6h, pH=7.5of reaction mediumconditions with the maximum immobilized efficiency of62.13%. After6consecutivecycles, the relative activity of immobilized enzyme retained about75%of its initial activity.Ionic strength have effect on the interaction between carrier and enzyme. The study showedthat the adsorption between carrier and enzyme is weakest when the concentration of NaClwas0.5mol/L.Enzymatic properties including heat stability, kinetic parameters of free andimmobilized lactase were compared. Compared with the free enzyme, suitable temperaturerange of immobilized enzyme shifted to higher temperature. It was found that immobilizedβ-galactosidase showed increased bonding affinity towards substrate with a smallerKm(1.44mmol/L) compared with the free one（3.18mmol/L）by Lineweaver-Burk method.Moreover, The storage stability of the immobilized lactase is stronger. After two weeks ofcold storage (4℃), the enzyme activity of immobilized enzyme remained more than75%ofinitial activity.Finally, immobilized K.fragilis β-galactosidase was successfully applicable in acolumn reactor for lactose hydrolysis and loading weight, reaction temperature andsubstrate flow rate on lactose hydrolysis rate were studied. The result suggested that lactosehydrolysis rate decreased with the increase of substrate flow rate and the maximum valuewas achieved at a4g/40ml loading weight,30℃. Choosing the optimal enzyme loading andtemperature conditions, with2ml/min substrate flow rate, lactose hydrolysis rate was64.39%. After eight continuous catalytic hydrolysis of the lactose, lactose hydrolysis rateremained above55%of the initial rate of hydrolysis. |
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