| As the catalyst of transphosphatidylation action of phospholipids, phospholipase D has been paid close attention for catalyzing and synthesizing various kinds of functional phospholipids product. Prepareing phospholipase D which has higher catalyzing activity, good operation performance and high preservation stability has become a research focus in the fields of phospholipids exploitation. In this paper, in the basis of the previous works, take the cross-linking as the core, phospholipase D was immobilized by Adsorption-Crosslinking, Adsorption- Aggregating- Cross-linking and Aggregating- Cross-linking. At the same time, the research of the operational conditions and performances of immobilized enzyme provide evidences for the research of enzyme immobilization methods and the applications of phospholipase D.The abstraction and purification of phospholipase D, fermented by streptomycete, which has been screened and conserved by the laboratory, has been studied in this paper. After the enzyme solution was purified by ammonium sulfate-alcohol coprecipitation percipitation, the result indicated, whose specific activity can reach to 10.45 U/mg, the purified factor is 11.32 and the activity recovery of immobilized PLDs has been gained to be 87%. This has supplied a satisfactory enzyme source for immobilization research.The combination of adsorption and cross-linking is the common method of executing carrier-bound immobilization on free enzymes. In this paper, as initial exploration, Adsorption-Cross-linking has been taken to immobilize PLD. The microcrystalline cellulose, diatomaceous earth and porous silica gel have been selected as carrier. The glutaraldehyde has been chosen as cross-linking agent. Diatomaceous earth has been found to be the best adsorption effect. The optimization immobilization conditions are as follows, the ratio of carriers to enzyme solution(specific activity:10.45 U/mg) are 1:4(g/mL), adsorption time is 3h, the concentration of glutaraldehyde is 1.5%, the cross-linking time is 3h. The activity recovery of immobilized PLDs has been gained to be 65% and the acthvity of immobilized PLD is 5.56U/mg in the optimization condition. However, this method has disadvantages of low enzyme immobilization recovery rate and the amount of carrier enzyme is hoped to boost.Based on the effect of precipitant, the free enzyme molecules will be assembled each other, reducing the solubility of the enzyme, and the carrier adsorption capacity of enzyme can be expected to increase. In this paper, the adsorption-crosslinking immobilization of enzyme has been strengthened by precipitating aggregated effect. A new method about the Adsorbption -Aggregation -Cross-linking immobilization has been proposed. The used precipitants were respectively ammonium sulfate, ethanol, acetone, n-propanol, isopropanol, methanol and PEG6000 and the crosslinking agent was glutaraldehyde. The impact of sedimentation on the stability of enzyme has been estimated by precipitation-re-dissolution method. The results showed that the loss activity of enzyme by ethanol induced has been the smallest. The activity recovery of enzyme using precipitation-dissolution method has reached 91%. Via single factor inspection and response surface analyze, the final immobilization conditions has been defined as follows:the carrier is diatomite, the pH of precipitant is 8.2, the saturation of ethanol is 81.6%, the concentration of glutaraldehyde is 2.87%, the cross-linking time is 4h and the ratio of carriers to enzyme solution(specific activity:10.45 U/mg) is 1:4(g/mg). The activity recovery rate of immobilized PLDs has reached 89%, the activity of immobilized PLD is 7.36U/mg. After reusing for 10 consecutive batches, the remaining activity of immobilized PLDs has been 60%. Clearly, the activity recovery and operational stability of immobilized enzyme by the new method have been elevated.In order to compare with the typical example method of Adsorption -Cross-linking, in this paper, carrier-free aggregating- cross-linking method has been explored to immobilizing PLD. Also, ethanol has been elected as precipitant and the cross-linking agent is glutaraldehyde. The optimal conditions for PLD-CLEAs have been as follows:the concentration of enzyme solution is 0.2g/mL(specific activity:9.5 U/mg), the saturation of ethanol is 72.6%, the concentration of glutaraldehyde is 0.22% and the cross-linking time is 1.8h. The activity recovery rate of immobilized PLDs can reached 86%, the activity of immobilized PLD is 15.62U/mg. Reuseing for 10 consecutive batches, the remaining activity of immobilized PLDs has been 55%. It has shown that this method also had a good effect on phospholipase D immobilization.Take the crosslinking as the core, assisting the precipitation to PLD immobilization that will implement carrier-free Aggregating-Cross-linking and Adsorbption-Aggregation-Cross-linking of PLD in the condition of higher activity recovery rate. The stability of immobilization enzyme is good.
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