Immobilization Of CALB Onto SBA-15 For Diacylglycerols Synthesis Through Glycerolysis

Diacylglycerols(DAG) have been recognized as functional cooking oil, due to their ability of reducing fat accumulation and therefore preventing obesity. Enzymatic preparation of DAG has some attractive features, such as mild reaction conditions, high selectivity and good efficiency. But the free enzyme is unstable and can not be reused. So it is difficult to be widely used in industry. However, taking advantages of the highly ordered mesostructure, large pores and abundant hydroxyl groups on the surface of mesoporous molecular sieve, the application of the mesoporous molecular sieve on enzyme immobilization has been attracted widespread attention. In this work, DAG was produced through glycerolysis with Candida antarctica Lipase B(CALB) immobilized on SBA-15 as catalyst, the main contents and results are as follows:(1) CALB was immobilized on SBA-15 through physical absorption, the immobilization conditions including p H, time and CALB concentration, were studied. The results showed that the optimum conditions for immobilization were as follows: the p H, immobilization time and concentration of CALB was 7.0, 30 min and 12 mg/ml(the concentration of protein is 70.2 μg/ml) respectively. Results from XRD, N2 adsorption-desorption and FT-IR spectra indicated that CALB was adsorbed into the channels of SBA-15 and the ordered mesoporous structure of SBA-15 after adsorption of CALB was retained. Meanwhile, the immobilizing efficiency and activity of CALB immobilized onto SBA-15 with different pore-diameters were studied. The results showed that, compared with the small pore-diameters, the larger one has a high activity but less immobilizing efficiency. The immobilized CALB showed better thermal stability than free CALB. After 4 h incubation at 60℃, 9.0% and 31.4% of the initial activity was respectively obtained from free CALB and immobilized CALB. The relative activity of immobilized CALB has been found to be 76.4% of its original activity after five reuses.(2) The CALB was immobilized on the mesoporous material SBA-15, and then used to catalyze the glycerolysis for DAG preparation. Through the means of single factor analysis, the optimum conditions are as follows: reaction temperature 50℃, 4.4g oil, 0.23 g glycerol, enzyme amount 5 wt% of substrate. Under these conditions, DAG content reached stable after 12 h reaction, whlie MAG content still incresed upon prolonging reaction time. The present SBA-CALB showed catalytic activity, in which nearly 60 wt% of DAG was observed, slightly higher than that from Novozym 435, in tert-amyl alcohol system. In the solvent-free system, the conversion rate of TAG is low because of low reaction rate. Meanwhile, the reusability of the SBA-CALB was evaluated and 95.8% of its initial activity was observed after five batch reactions.(3) Mesoporous silica SBA-15 was modified by imidazole-based ionic liquids with alkyl groups. The modified SBA-15 was then used to immobilize CALB and the obtained catalysts were used to catalyze the glycerolysis for DAG preparation. Compared with the immobilized lipase on parent SBA-15, the immobilized lipases on modified support samples were less sensitive to temperature and showed higher specific activity. Of the modified SBA-15 samples, methyl-functionalized ionic liquid modified SBA-15 afforded the highest activity for CALB, which improve 2.7 folds compared with CALB immobilized on SBA-15. In the glycerolysis for DAG preparation, the conditions are as follows: reaction temperature 50℃, reaction time 12 h, 4.4g oil, 0.23 g glycerol, tert-amyl alcohol 16.3ml and 5 wt% of CH3-IL-SBA-CALB(based on substrate) amount. 71.5% of TAG conversion and 67.1 wt% of DAG were obtained, which are markedly higher than that from SBA-CALB.