Preparation and characterization of immobilized phospholipase A 2 on chitosan beads for lowering serum cholesterol concentration

Abstract

Phospholipase A 2 (PLA 2) from cobra venom, which can hydrolyze the S N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10 −3 to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA 2 was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA 2 could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA 2 should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.