Characterizations Of Immobilized Earthworm Fibrinolytic Enzymes On Magnetic Chitosan Microspheres

Earthworm fibrinolytic enzymes(EFES)are a group of proteases with both plasmin and plasminogen activator activity,which were isolated from earthworm.As a potential thrombolytic agent,they have anticoagulant,dissolving fibrin,supporting healthy blood circulation properties.In this paper,magnetic chitosan microspheres with epoxy-activating and coupling soybean trypsin inhibitor were used as a carrier to immobilize EFES,aiming to provide a theoretical basis for the separation and purification of EFES.Details as follows:Magnetic chitosan microspheres were prepared with Fe3O4,glutaraldehyde and span-80 via emulsion cross-linking method.The optimal conditions was identified to be:concentration of chitosan was 10 mg·mL-1,oil-water ratio was 5:1,agitation rate was 600 rpm,volume fraction of glutaraldehyde was 3%.The physical properties of the microspheres were characterized by FTIR,SEM and VEM,the results showed that the microspheres have uniformly distributed granular diameters and the average diameter was 215 nm,Fe3O4 was successfully wrapped up in the microspheres.Magnetic analysis showed that the saturation magnetization of microspheres was 66.84 emu·g-1 with super paramagnetic.Magnetic chitosan microspheres was activated with epichlorohydrin,the optimal activation conditions as follows:DMSO concentration was 50%(v/v),epichorohydrin volume fraction was 40%(v/v),concentration of NaOH was 1.6 mol·L-1,reaction time was 4 h,activation temperature was 40?.The density of epoxy group was 211 ?mol·g-1.Soybean trypsin inhibitor(STIB)was coupled to the microspheres modified with epoxy,the optimal conditions for coupling have been obtained:pH was 7,coupling time was 6 h,vibration speed was 200 r·min-1,content of soybean trypsin inhibitor was 16%.The coupling rate could reach 13.69mg·g-1.Magnetic chitosan microspheres coupled with STIB was used to EFES immobilization,the optimal conditions was identified to be:immobilization was 2.5 h,pH was 7.5,solid-to-liquid ratio was 1:20,enzyme concentration was 15 KU mL-1.On the best immobilized conditions the immobilized enzyme activity and the dynamic recovery were 166 U·mg-1 and 55%,respectively.Moreover,enzymatic properties was also studied,the results showed that immobilized enzyme and free enzyme have similar enzymatic characteristics,but the former exhibited enhanced pH stability,storage stability,it still retained more than 78%of its initial activity after storaged for 30 d under 4? while free enzyme only retained 32%.Relative activity of immobilized and free earthworm fibrinolytic enzyme gradually decreased and the rate of enzymes rapidly inactivated with the increasing of temperature,both free and immobilized enzyme activity were decreased seriously when up to 60?,but the latter showed better thermal stability.Enzymatic kinetics analysis indicated that the apparent activation energy of immobilized and free earthworm fibrinolytic enzyme were 130.43 kJ·mol-1 and 116.65kJ·mol-1 respectively between 30? and 60?.