Studies On The Immobilization Of Permeabilized Flavobacterium Heparinum And Its Degradation Of Heparin

Heparin belongs to the family of glycosaminoglycans(GAGs),the molecule is composed of glucosamine,uronic acid and their sulfated and acetylizad derivatives, which connect withβ-1,4 glucosidic bond and form the linear chain polysaccharides. Low molecular weight heparin(LMWH) is the product of depolymerised heparin according to different degradation methods.Compared with heparin,LMWH is characterized by stronger selectivity and antithrombotic capability,with extensive perspective of application in clinic.Different degradation procedures produce oligosaccharides with different structures,resulting in different bioactivity diversities. Enzymatical preparation of LMWH has characteristics of mild reaction condition, clear enzyme cutting site,no changing of heparin structure and no incorporation of extraneous material.Heparinase can split the glucosidic bond of heparin master strand specifically, which is initially found and isolated from Flavobacterium heparinum.As its stability is poor,the purification of heparinase results in great loss of enzyme activity. Immobilization of enzymes can overcome the problems of poor stability,easy deactivation and inability of reuse of the enzyme.This paper reports the permeabilizations and immobilizations of two strains of Flavobacterium heparinum.The optimum condition of immobilization,physicochemical properties of immobilized cells,degradation conditions and properties of products were analyzed and determined,indicating that immobilized cells possess better operation and storage stability.Under proper degradation conditions,LMWH with uniform molecular weight and fine anticoagulation activities were produced.All the results of experiment were summarized as follows.(1) Establishment of enzymatic activity determination system of heparinase Standard curve of heparin content was installed by Azure A method,and enzymatic determination systems of crude heparinase preparations,permeabilized Flavobacterium heparinum cells and immobilized Flavobacterium heparinum cells were established.Enzymatic determination was carried out throughout all the experiment phases to calculate recovery rate and stability exactly,which were significant indexes of permeabilization and immobilization.(2) Determination of cultivation and induction conditions of Flavobacterium heparinum Two strains of Flavobacterium heparinum were inoculated to DSMZ medium and NBRC medium,and growth curves were determined.NBRC medium was chosen for the following experiments.Flavobacterium heparinum strains were cultured in 30℃at the speed of 200r/min with rocking bed for a certain time when the OD600 reached about 1.0 and then 1 mL sterile heparin with the concentration of 0.1 g/mL was added to induce the production of heparinase.Full-scale verification methods based different mechanism of Flavobacterium heparinum including the use of preliminary screening medium,test under microscope,heparinase enzymatic activity determination and PCR of HepA were established.Crude heparinase preparations were obtained by supersonic cell breakage and high pressure homogenization,and the latter was chosen for the following experiment after comparing the effects of the two methods.The operation stability and storage stability of crude heparinase preparations were poor,with less than 10%activity maintained after 5 reactions and 35%activity left after storing at 4℃for 120 h.(3) Permeabilization of Flavobacterium heparinum Flavobacterium heparinum strains were permeabilized by Triton X-100,SDS,CTAB,EDTA,Tween 80 and lysozyme respectively,and operating complexities as well as effects were taken into consideration.Lysozyme of 100mg/L was chosen as permeabilizing agent that could maintain as high as 50%of heparinase activity after the enzyme treatment.The stability of permeabilized ceils was higher than that of crude haparinase preparations with more than 40%activity left after 10 reactions.(4) Immobilization of permeabilized Flavobacterium heparinum cells Permeabilized Flavobacterium heparinum cells were immobilized by embedding method with calcium alginate,and the optimization condition of immobilization determined by orthogonal experiment was 1.5%sodium alginate,5%CaCl₂ and 4 h hardening at 4℃.Physico-chemical properties of immobilized cells including optimal temperature,stability of temperature,optimal pH and stability of pH were studied. The optimal temperature was 30℃,and the optimal pH was 7.0.Compared with crude heparinase preparations,immobilized cells had higher stability and broader optimal temperature and pH.The heparinase activity recovery rate of immobilized cells was about 30%.And their stability was much higher than that of permeabilized cells with more than 60%activity left after 10 reactions and 90%activity left after storing at 4℃for 120 h,displaying satisfactory perspective of application.(5) Preparation of LMWH by immobilized Flavobacterium heparinum Three degradation reactions for different treatment time by immobilized cells were studied, and three samples of LMWH OH-1,OH-2 and OH-3 were obtained by ultrafiltration, concentration and freeze drying.All the samples had absorption maximum at 232 nm. GPC-MALLS was used to determine their molecular weight.The average molecular weights of OH-1,OH-2 and OH-3 were 8798 D,6709 D and 5671 D and the polydispersities of the three samples were 1.375,1.383 and 1.408 respectivel.;The anti-coagulation potencies of OH-1,OH-2 and OH-3 were 26 USPU/mg,15 USPU/mg and 11 USPU/mg,the anti-Fxa activity were 44 IU/mg,36 IU/mg and 32 IU/mg respectively,and the ratios of anti-FXa activity and anti-coagulation potency all exceeded 1.5.