The Immobilization Methods And Applications Of Acid Urease

The urea content in wine could be reduced by adding acid urease, however, it is difficult to separate and reuse the free urease in wine. In fact, immobilized enzyme can avoid these deficiencies, which can expand the field of use of the enzyme.The chitosan was chosen as the main carrier, and two methods were established to immobilize acid urease, one is based on chitosan-gelatin composite, and the other is based on chitosan nanoparticles. For chitosan-gelatin composite-based method, the optimal conditions for preparation of immobilized enzyme membrane were as follow: the membrane was made of 1.0% of chitosan and 0.75% of gelatin, and then urease was crosslinked with 0.02% glutaraldehyde. The optimum immobilization time was 2 h, with the recovery rate of 69.1%. Compared with free enzyme, immobilized enzyme has better pH and thermal stabilities, longer storage half-life up to 81 days. Even after 10 times repeated measurements, only 9% of enzyme activity lost. For chitosan nanoparticles-based method, the optimal conditions for immobilization were: chitosan: sodium polyphosphate was 5:3, the volume of enzyme was 1.5 mL, andthe enzyme activity recovery of 81.7% was obtained. The urea removal rate reached 52.7% after continuous treatment of 1 L wine.From the consideration of enzyme activity recovery rate, preparation procedures, re-usability and thier applications, chitosan-gelatin composite film was chosen to immobilize acid urease.In order to improve the stability of acid urease, the effects of sugars, polyols, inorganic salts and other types of stabilizers were tested by adding them to the immobilized enzyme membrane respectively. Based on the orthogonal experiments, the optimal combination for protective reagent was listed as follows: sucrose (3.0 g/L), glycerol (15%v/v), and NaCl (15 mmol/L). By using the protective reagent, the enzyme activity (unit per area) was increased to 0.68 U/cm2, with extended storage half-life of 100 days. After addition of protective reagent, the usage half-life was significantly improved, and two more sample precessing cycles can be operated compared with those of without stabilizer.The acid urease enzyme membrane was made as enzyme membrane reactor in the form of roll film. After 70 hrs continuously dealing with 2 L wine at the flow rate of 0.5 mL/min, , the urea removal rate in rice wine was 54.8%, and the content of ethyl carbamate was reduced to 55.6%. In addition, the flavor of wine and the content of polyphenols in rice wine remain unchanged.Chitosan-gelatin composite was further applied to co-immobilize acid protease and papain to hydrolyze soy protein. 32% of hydrolysis degree could be achieved. In the hydrolytic product of co-immobilized enzymes, the amino nitrogen content was increased 1.84 times, the total free amino acid content was increased 2.12 times. The molecular weight of poly-peptides in non-enzymatic hydrolytic soy protein was mainly consisted of small peptides.As shown in our results, the chitosan-gelatin-based immobilization strategy is not only suitable for the immobilization of acid urease and similar enzymes, but also for immobilization applications of enzymes which have a wider range of optimum pH in the acid range.