Immobilization of phospholipase D on macroporous SiO2/cationic polymer nano-composited support for the highly efficient synthesis of phosphatidylserine.

Novel nano-composites were prepared by coating epoxy resin-based cationic polymer in nano-thickness via in-situ curing on the nano-wall of macroporous SiO2 with pore size of 0.5∼1 µm. By changing the thickness of polymer coating the specific surface area and porosity varied in range of 115∼74 m2/g and 90.4∼83.9 %, respectively. Through ion exchange phospholipase D (PLD, from Streptomyces sp) was efficiently immobilized on the nano-composites as support and the immobilized PLD was applied for the highly efficient synthesis of phosphatidylserine (PS). The loading amount of PLD on the nano-composited support reached to a maximum of 90.2 mg/gsupport, 4 times as high as that on the pure macroporous silica. The specific activity of the immobilized PLD reached as high as 16,230 U/gprotein, while that of free PLD was 18,780 U/gprotein. Under a wide range of temperature and pH the stability and activity of the immobilized PLD were greatly improved as compared with the free ones. Under optimized conditions at 45 °C and pH 7.0, the PS yield reached as high as 96.2 % within 40 min. After 28 days storage the immobilized PLD retained 82.2 % of original activity, and after 12 cycling reuses it retained 79.3 % of PS yield, which indicated that the immobilized PLD exhibited good stability.

Efficient immobilization of phospholipase D on novel polymer supports with hierarchical pore structures.

In this article, novel epoxy resin-based hierarchical porous polymers (HPSs) have been prepared through a non-sol-gel and template-free approach using crystalline trimethylolpropane (TMP) as porogen. The polymers exhibit dimensional stability and possess 3-dimentional interconnected multi-scale pores. In range of 50 µm~10 nm are ultra-macro-pore in between skeleton, macro-pore on skeleton and meso-pore in network, respectively. The porosity and specific surface area can be adjusted in range of 91.2-82.5% and 225-156 m2/g, respectively. Using three kinds of hierarchical porous polymers as supports phospholipase D (PLD) was effectively immobilized through physical adsorption. Owing to high porosity of the support and improvement of mass transfer the loading amount of PLD reached as high as 223 mg/gsupport and the corresponding specific activity achieved up to 3.75 × 103 U/gsupport. Under optimized conditions and the phosphatidylserine (PS) yield reached 95.5% within 40 min at 45 °C. The immobilized PLD exhibited not only better storage stability and but also resistance to pH and thermal inactivation than free PLD. It was found that 73.5% of PS yield retained after 12 cycling reuses.