Preliminary Study On The Immobilization Of Thermostable Enzymes

Thermostable enzyme is a general name of enzymes which can still maintain a certain activity under high temperature. Thermostable enzymes are not only far superior to chemical catalyst in catalytic efficiency and substrate specificity, but have excellent stability under the condition of high temperature as well. As a consequence, it can overcome the unstability in biological property exhibited by medium-temperature enzyme (20℃~ 55℃) and low-temperature enzyme (-2℃~ 20℃) , which make many high-temperature chemical reaction process realizable. Free enzyme can be used only once, on the contrary, immobilized enzyme used as biocatalyst can be utilized repeatedly, and what is more, it has the advantage of stable activity, simple operation and facility to separation and purification for production. Thus, it is significant to research the immobilization.In this thesis, vector particles were discussed firstly, the effect of preparation conditions on the properties of particles was also inspected. On the basis of the above research, two types of gel particles, calcium alginate gel particles and particles of polyvinyl alcohol gel were prepared. Through the comparison between the performances of the two gel particles, we choose calcium alginate gel particles as carrier.Immobilized glucose isomerase was prepared using entrapment-crosslinking, the optimum condition obtained from single factor experiment was: the volume ratio of enzyme solution to sodium alginate was 1:2, glutaraldehyde concentration was 0.01%, gel concentration was 30 g / L, CaCl2 concentration was 0.2 mol / L, immobilization time was 6 h. Through the experiment of immobilized enzyme protein in different temperatures, it is inferred that the protein leakage is the main source of change influenced by temperature during the use of immobilized particles.Resins with various function groups were tested. The immobilized enzyme prepared by absorption-crosslinking with a carrier of D380 was selected. With the research and optimize on the addition amount of enzyme, the concentration of crosslingking agent, the absorption time and the absorption pH, etc. The optimum condition ofα- amylase immobilization was: glutaraldehyde concentration was 0.005%, treatment time was 45 min, concentration of enzyme was 3 mg/g (protein/vector), the enzyme solution pH was 5.8, absorption temperature was 25℃, absorption time was 6~10 hour, and the obtained immobilized enzyme activity was 80 U / g. The optimum condition of Glucose isomerase immobilization was: glutaraldehyde concentration was 0.01%, the treatment time was 45 min, the concentration of enzyme was 10 mg/g (protein/vector), the enzyme solution pH was 7.0, the absorption temperature was 30℃, the absorption time was 8 hour and the obtained immobilized enzyme activity was 600 U / g.The enzymatic property of immobilized glucose isomerase was researched. The enzyme's optimum temperature was 90℃and the optimum pH was 7.0. The pH stability and thermal stability was far superior to that of free enzyme. Immobilized enzyme's apparent Km was 602 mmol / L, which was higher than that of free enzyme. The effect of different substrate concentration on the conversion rate of glucose was studied. Immobilized enzyme repeatedly utilized for six times had a conversion rate of glucose remaining at 50%.