Efficient synthesis of N-acetyllactosamine using immobilized ß-galactosidase on a novel 3D polymer support.

A novel polymer support was prepared by curing of epoxy resin in ethanol solution in the macropores of a melamine sponge. The produced polymer gel could uniformly deposit on the surface of melamine in either porous or nonporous morphology. The composite sponge with porous coating can be used as a large-sized and well-mass transferred support for the immobilization of ß-galactosidase from Bacillus circulans through method of adsorption and crosslinking, and a column reactor was made for the preparation of N-acetyllactosamine in a sealed circulation way. The porosity and specific surface area of the support were 91.6 % and 46 m2/g, respectively. The loading amount and the specific activity of immobilized ß-galactosidase under the optimal immobilization conditions were 41.2 mg/gsupport and 16.5 U/mgprotein, respectively. In the biosynthesis of N-acetyllactosamine lactose and N-acetylglucosamine were used as donor and acceptor, respectively. Under optimized conditions the N-acetyllactosamine yield reached 54 % within 150 min at 50 °C. After 10 cycles, the immobilized ß-galactosidase retained 70 % of the original activity.

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.

Highly active and stable nanobiocatalyst based on in-situ cross-linking of phospholipase D for the synthesis of phosphatidylserine.

Phospholipase D (PLD) was effectively immobilized on a ZnO nanowires/macroporous SiO2 composite support through an in-situ cross-linking method. An anionic and long-chained bi-epoxy cross-linker was used by adsorbing on the surface of ZnO nanowires through static interaction before cross-linking. Under the fine control of in-situ cross-linking the immobilized PLD has loading amount as high as 113.7 mg/gsupport, possessing high specific activity from 13,987 to 16,142 U/gprotein in all the range of loading amount. The immobilized PLD showed high activity and stability in catalyzing the conversion of phosphatidylcholine (PC) to phosphatidylserine (PS). The reaction conditions such as loading amount of PLD, substrate molar ratio, temperature, solution pH, and reaction time were optimized for the finding of best synthetic process. Under optimized conditions and the PS yield reached 94.8% within 40 min at 50 °C. The immobilized PLD exhibited not only better thermostability and resistance to pH inactivation than free PLD but also the greatly improved storage stability and reusability. It was found that 81.5% of initial activity retained after incubation at 4 °C for 60 days and that 80.4% of PS yield retained after 13 cycling reuses.