The Immobilization Of Acid Urease And Preliminary Study Of Enzyme Reactor

Acid urease can decompose urea, which is a harmful substances in Chinese rice wine,ultimately limiting the content of carcinogenic ethyl carbamate in wine. In this paper,immobilized acid urease with carrier and non-carrier were both adopted. The characteristics ofthe immobilized enzymes were studied and their applications in rice wine were analyzed.Urease from Providencia sp. JNB815was separated and purified by ethanol fractionation,DEAE ion exchange, Resource Q ion exchange and Superdex200gel filtrationchromatography. In each purification step, the activity of urease and urethanase wasdetermined. The purified enzyme performed activity to both urethane and urea. It was initiallyspeculated that the enzyme exhibited activity of bifunctional enzyme. Acid urease andurethane degrading enzyme yield were38.3%and23.6%, respectively. The purification factorreached5.0and3.1.By using a compound method of adsorption, embedment and crosslink, the bifunctionalenzyme produced by Providencia sp. JNB815with both activity of urease and urethanase wasimmobilized. With urease activity as an indicator, the optimum immobilization conditionsfrom single factor experiments were as follows: chitosan concentration4.0%and gelatinconcentration2.0%. The mixture of them was dropped into condensation solution to formmicrospheres. Crosslink of the microspheres was conducted with0.4%of genipin as thecrosslinker and kept at30℃with concussion for6h. Then the urease was immobilized to thecrosslinked microspheres for8h.The optimum pH was4.0and the optimum temperature was40℃when the immobilizedenzyme catalyzed urea. While urethane was as the substrate, the optimum pH was4.5, and theoptimum temperature was60℃. The immobilized urease maintained stability when thetemperature was in the range of20~50℃and pH ranged from4.0to6.5. The immobilizedurethanase maintained stability when the temperature was in the range of20~60℃and pH5.0to6.5. It retained80%and30%of the original catalytic avtivity to urea and urethane,respectively after being used for10times, which performed good reusability. The Kmof theenzyme immobilized by chitosan microspheres and free enzyme with urea as substrate was13.54mmol·L-1and5.99mmol·L-1, respectively. When urethane was as the substrate, the Kmwas705.78mmol·L-1and183.82mmol·L-1, respectively. The higher Kmof the immobilizedenzyme suggested that its affinity to the substrates became weaker.Under the condition of32℃and100r·min-1,0.04U·mL-1of immobilized urease wasadded into the Chinese rice wine. After processing for20h, the removing rate of urea in winereached93.03%. In the same condition, when the amount of urethanase added was0.17U·mL-1, the removing rate of urethane in wine was56.60%after20h. Furthermore, thetreatment with immobilized enzyme showed neglectable effect on the volatile flavorsubstances in the wine.Conditions of crosslinked urease aggregates were: genipin concentration0.3%andcrosslinked for2.5h, with0.3g·L-1bovine serum albumin in auxiliary. The Kmof CLUAswith urea and EC as substrates was5.65mmol·L-1and627.15mmol·L-1, respectively. Removal rate of urea was up to85.35%when crosslinked urease aggregates were initiallyused. After reusing the crosslinked urease aggregates for six times, the removal rate of ureawas still up to69.30%. When the cross-linked urease aggregates were applied in a batchstirred and membrane reactor, the removal rate of urea in wine was80.49%and the removalrate of urethane was46.27%. This treatment compounded no significant change of volatileflavor substances in wine.