Study On The Immobilization Of α-acetolactate Decarboxylase And Its Application

Diacetyl is a by-product of beer fermentation. When its concentration is higher than 0.15 mg/L, it will result in a very unpleasant taste, like sour food taste. In traditional brewing, green beer needs three to six weeks storage at low temperature to reduce the content of diacetyl. Alpha-acetolactate decarboxylase (EC.4.1.1.5, ALDC) could quickly transfer theα-acetolactate to the acetoin, and accelerate the beer to mature.In this article, immobilization of ALDC by industrial resins was studied. The optimal conditions of enzyme immobilization were mainly investigated by the single factor and the orthogonal experiments. The basic enzymatic properties of immobilized ALDC and free enzyme were researched. Immobilized ALDC was used in continuous beer fermentation. The effects of filling amount of immobilized enzyme, fermentation temperature and retention time on the reducing rate of diacetyl were studied. Main results are as follows.(1) Through primarily researches on seven kinds of resins, the immobilized enzyme was prepared by absorption with a carrier of D314FD(2) The effects of the amount of ALDC, adsorption time, adsorption temperature and pH on the activity and efficiency of immobilized enzyme were investigated. The optimal conditions for the immobilization were as follows, 6.25 mL liquid ALDC was added into one gram of pretreated resin, stirred for 10 hours at 15℃in buffer of pH 7.5. Under the optimal conditions, the immobilization efficiency could reach to 44%.(3) Compared with free ALDC, the characteristics of immobilized enzyme were as follows, the optimal temperature was 40℃whereas the free enzyme was 35℃. The optimum pH was 5.5 whereas the free enzyme was 6.0. Compared with the free enzyme, immobilized ALDC has higher thermal stability, broader pH stability and better reusability.(4) Immobilized ALDC was used in continuous beer fermentation. The results showed that the filling amount of immobilized enzyme was 45 g in the volume of 120 mL glass column , the?reaction temperature was 11℃, the retention time was 14 h. Under this optimal condition, the reducing rate of diacetyl could reach to 43.5%, and the content of diacetyl is 0.095 mg/L. The average reducing rate of diacetyl was 33% after 30 days continuous operation. It indicated that the immobilized enzyme had good operation stability.?...