Preparation Of The Immobilized Lactase And Its Application

Lactose was catalyzed by lactase to perform hydrolysis reaction and transglactosylaction reaction. It was hydrolyzed to form glucose and galactose at low concentration and to form galactooligosaccharides in transglactosylation reaction at high concentration. So, lactase could be applied in the preparation of low-lactose milk and galactooligosaccharides. Low-lactose milk played an important role in satisfying great need to dairy from lactose-intolerance and enhancing the development of dairy industry in China. Galactooligosaccharides was a kind of new food additive with good health benefits. It had large consumption market. From these views, lactase was the key.Enzyme was restricted to certain room in immobilized enzyme compared with the free enzyme. The catalyst could be call backed and recycled. It took great advantages in resource and energy sources economized, cost economized, environment protected, production automatised and continued.A method to prepare the immobilized lactase was studied in the dissertation. The lactase was an extra-cellular product secreted by Pichia pastoris constructed by Chinese Academy of Agricultural Science. The immobilization method was a combination of absorption-entrap-crosslink and the target to reuse of the enzyme was achieved. The immobilization process was optimized. After the properties of the immobilized and free lactase were compared, significant changes were observed. In the respect of the optimum temperature, the free enzyme was 65℃while the immobilized enzyme decreased markedly to 35℃. In the respect of optimum pH, the immobilized enzyme was pH 2.5, a little lower than that of the free enzyme (pH 3.5). The immobilized and free lactase showed 80% or higher activity during the range of pH 2.5-6.0 and pH 3.0-6.0, respectively. The kinetic constants also changed much. For the immobilized and free lactase, Km, Vmax were 0.714 mol/L, 626.56μmol/ min·mL and 0.267 mol/L, 666.67μmol/ min·mL.The operational stability of the immobilized lactase was studied in the paper in respect of lactose hydrolysis, low-lactose milk and galactooligosaccharides (GOS) preparation.With 5% lactose water solution as substrate, lactose hydrolysis maintained a level of 75% after 52 cycles in batch operation. Lactose hydrolysis kept a level of 80% after 62 d continuous operation. Its operational half life was 136 d.Low-lactose milk was prepared by the immobilized lactase in the packed-bed reactor. When dilution rate was controlled at 0.5 h^-1, lactose hydrolysis achieved 70%. In the 30 d continuous operation, the residual lactose below 15 g/L was maintained.The immobilized lactase was applied in GOS production. The conditions were optimized in batch operation. They were that the temperature was controlled at 35℃, pH 6.5-7.0 and the lactose concentration was 40%. The maximum value of GOS was 160 g/L. After 7 recycles, the immobilized lactase still showed good operational stability. Effect of dilution rate on the formation of GOS was studied in the packed-bed reactor. When dilution rate got 0.5 h^-1, the maximum value of GOS concentration (148 g/L) was achieved. When dilution rate got...