| Phospholipase D (PLD), which is also called as phosphatidylcholinase [EC3.1.4.4], plays an important role in catalysing organic alkali group exchange reactions of phospholipids. This kind of reactions can be applied in the phospholipid structure transformation, which is a crucial technology in preparing various semi-sythetic phospholipids. However, there are some difficulties in PLD production, such as low fermentative activity and purification problems. Besides, binary-water phase system is necessary to catalytical reactions of phospholipids. Hence, PLD immobilization calls for more advanced technologies. Covalent immobilization is a promising method, which combines the enzyme and the carrier by covalent bond with a good stability and repetitiveness. In this paper, the study of PLD immobilization was investigated in detail.The PLD immobilization was carried out by the combination with an actived cellulose carrier, which was actived by epichlorohydrin (ECH). The alkli type and concentration, the ratio between cellulose and ECH, as well as the actived time, which affected the efficiency of PLD immobilization, were invertigaed in this paper. The optimal condition of carrier activation was: 1 mol·L-1 NaOH 20 ml, microcrystalline cellulose 1 g, ECH 4 ml. Further more, immobilization time, temperature, the ratio between carrier and enzyme were also inverstigated. The optimal condition of PLD immobilization was: epoxy cellulose 1 g, PLD solution 30 ml (crude protein 0.8 g·L-1) and the immobilization temperature and time were 37℃and 12 h respectively. In this condition, the immobilization efficiency reached to 48.8%. Compared with the free PLD, the immobilized one had the higher thermal and operational stabilities.In this section, we took glucoamylase in stead of PLD for lower cost. Cellulose, coarse pore silica gel and epoxy-cellulose were taken as enzyme carrier in glucoamylase immobilization. The results showed that the protein loading efficiency can be improved by adding precipitant regardless of carrier types. The PLD immobilization adopted an enrich cross-link method with the cellulose carrier. The optimal condition was as following: the ratio among enzyme solution, carrier and percipitant was 5ml·0.8mg/ml: 1g: 10ml (NH4)2SO4. After absorption of enzyme and the carrier, precipitant and glutaraldehyde were added in to the system at the same time. The initial concentration of glutaraldehyde was 0.12%. The reaction processed by magnetic stirring for 40 min and then added more glutaraldehyde to reach to a concentration of 0.18%. Further reaction was carried out for 30 min in the shaker. At last, immobilized enzyme was acquired after filtering, washing and vacuum. The immobilized enzyme by enrich cross-linking method was very stable and could be preserved for a long time, which was similar with the characters of enzyme immobilized by covalent binding. The immobilized enzyme acquired by this method is difficult to deactive and be affected by mass transfer, which facilitates quality control.
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