Immobilized Technique Of Lactase And Application In Low-lactose Milk

For making lactase from Aspergillus niger be suitable for hydrolyzing the lactose of milk and achieving the continuous production of low-lactose milk, surrounding with theories and techniques related to immobilization of lactase and production of low-lactose milk by immobilized enzyme, the paper systematically studied the selection of carrier, optimization of immobilization, properties of immobilized enzyme and continuous production of low-lactose milk. The main results were as follows:1. Developing an ideal carrier is one of the important topics of immobilization study. Kation-exchange resin D151 was selected from four kinds of resins as good carrier to absorb lactase from Aspergillus niger. The optimal conditions of absorption and crosslinking were studied with glutaraldehyde as crosslinking agent and Kation-exchange resin D151 as carrier. After having been diluted with pH4.0 0.3 mol/L acetic acid buffer, lactase was absorbed on resin D151 with comparison of 50 U enzyme per g wet resin for 24 h at 25℃, then lactase was crosslinked with 4% glutaraldehyde for 6 h at 30℃. The enzyme activity reached 11.8U/g(carrier) and the activity yield was 37.2%.2. The test of immobilized enzyme indicated that the optimal temperature of the immobilized enzyme was 60℃, which was lower than that of the soluble enzyme by 10℃. Thermal stability of the lactase was reduced by immobilizing treatment. The optimal pH of lactase removed to alkalinity by immobilization. There was great difference between the pH stability of soluble and immobilized enzyme. Compared with the soluble enzyme, the immobilized enzyme worked better under the condition of pH6.5. The K' m value of immobilized enzyme was 132.1 mmol/L for lactose, which was a little higher than soluble enzyme. The metal ions Na⁺, Ca²⁺, Cu²⁺, Zn²⁺ had different inhibition on soluble and immobilized enzyme. Pb²⁺, Mg²⁺ had inhibition on soluble enzyme, but they had activation on immobilized enzyme in which the effect of Mg²⁺ was the most obvious. K⁺, Fe²⁺, EDTA had no inhibition and activation on soluble and immobilized enzyme. The storage stability in 4℃ refrigerator was improved after immobilization. The remain activity was above 90% after the immobilized enzyme was repeatly used 10 times under the condition of using pH4.0 0.2 mol/L acetic acid buffer to wash immobilized enzyme for 10 minutes after reaction. Under the condition of pH6.5, 50℃, the half-life of the soluble enzyme was only 9 days, while immobilized enzyme reached 24 days.3. Through the research on the conditions and the usage stability of productionlow-lactose milk by immobilized enzyme, the results indicated: the result of continuous production low-lactose milk in the filled-bed reactor was the best in the space velocity of 1.5 h'1 at 50 °C (practical velocity 0.53 mL/min^ staying time 40 min) and the hydrolysis rate of lactose reached 79.7% which answered for the request of low-lactose milk.After continuous operation of the column packed with immobilized enzyme for 10 days in the space velocity of 1.5 h'1 at 50°C, 12% of initial enzyme activity was lost under the condition of using pH6.5 buffer to wash column per 20 h and the hydro -sis rate of lactose was 70.1 % which accorded with the request of low-lactose milk. The half-life of immobilized enzyme was about 22 days.