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.

Improved biodegradation of synthetic azo dye by horseradish peroxidase cross-linked on nano-composite support.

A ZnO nanowires/macroporous SiO2 composite was used as support to immobilize horseradish peroxidase (HRP) by in-situ cross-linking method. Using diethylene glycol diglycidyl ether (DDE) as a long-chained cross-linker, it was adsorbed on the surface of ZnO nanowires before reaction with HRPs, the resulted composite was quite different from the traditional cross-linking enzyme aggregates (CLEAs) on both structure and catalytic performance. The immobilized HRP showed high activity in the decolorization of azo dyes. The effect of various conditions such as the loading amount of HRP, solution pH, temperature, contact time and concentration of dye were optimized on the decolorization. The decolorization percentage of Acid Blue 113 and Acid black 10 BX reached as high as 95.4% and 90.3%, respectively. The immobilized HRP gave the highest decolorization rate under dye concentration as 50mg/L and reaction time of 35min. The immobilized HRP exhibited much better resistance to temperature and pH inactivation than free HRP. The storage stability and reusability were greatly improved through the immobilization, from the decolorization of Acid blue 113 it was found that 80.4% of initial efficiency retained after incubation at 4°C for 60 days, and that 79.4% of decolorization efficiency retained after 12 cycles reuse.