Preparation Of New Immobilized Lipase And Its Transesterification Activity

Lipase as a biological catalyst has properties with high selectivity, mild conditions of catalytic reaction and no pollution. The immobilized lipase has become so important for study and application because they can overcome the most of disadvantages of natural enzymes including weak stability, easy inactivation, none re-use, mixed with reaction products, and difficult purification.In the present work, the immobilization method of lipase on cotton towel as supporting material, PEI as adsorbent and GA or THP as crosslinking agent were incvestigated systematically. Evaluation of transesterification activity of immobilized lipase was carried out. The main results were shown as follows.1. The influence of solutions concentration, pH, temperature in the process on the effect of lipase immobilization was studied. The optimal conditions for preparation were obtained by single factor experiment: The concentration of PEI solution was 3.0 mg/ml at pH 7.0; the concentration of GA solution was 0.2% at pH 8.0; the temperature for crosslinking was 40℃; the time for crosslinking was 60 min.2. According to the single-factor experiment, PEI concentration and pH, GA concentration and pH, crosslinking time and temperature were confirmed. So, three levels of the factors including PEI concentration, GA concentration and the crosslinking time were selected for the respond surface analysis. The results showed that the optimum immobilized conditions were obtained as follow: PEI concentration 3.37mg/ml, GA concentration 0.26%(v/v), crosslinking time 50min, which led to estimated and observed values of maximal transesterification activity of 5.03U/g immobilized lipase and 4.99U/g immobilized lipase, respectively.3. The influence of buffer solution pH to the activity of immobilized lipase was optimized and found: The optimal pH of buffer solution of lipase from Porcine pancre was 4.5 and the activity of immobilized lipase was 5.02U/g immobilized lipase; The optimal pH of buffer solution of lipase of esterification was 5.0 and the activity of immobilized lipase was 12.45U/g immobilized lipase; The optimal pH of buffer solution of Alkaline lipase was 9.0 and the activity of immobilized lipase was 9.81U/g immobilized lipase. 4. The lipase was immobilized on cotton towel with a novel coupling reagent tris(hydroxymethyl) phosphine (THP) and a novel adsorbent polyethylenimine (PEI). The optimum condition of immobilization was investigated, and the result showed that: The quantity of PEI was 0.5ml (the concentration of PEI was 3.37mg/ml, pH was 7.0), the quantity of THP was 2ml (the concentration of THP was 0.10% (v/v)) every 0.2g cotton towel. The temperature of immobilization was 40℃, crosslinking time was 20min. The experiment showed that THP-immobilized lipase catalytic transesterification of vinyl butyric acid and Pentanol, the conversion rate in 1h was 92.1%, the activity of immobilized lipase was 15.80U/g immobilized lipase. Compared with the GA-immobilized lipase, the THP-immobilized lipase had higher reusability, thermal stability and activity than that immobilized by GA. This was a cheap and readily available new method of immobilization of lipase.5. The influence of proportion of THP and GA to the activity of transesterification was studied. The transesterification activity of immobilized lipase was 28.17U/g immobilized lipase when the ratio of GA and THP was 1:1.