Immobilization Of Alkaline Protease And Preparation Of Anti-osteoporosis Fish Skin Collagen Active Polypeptide

Fish skin collagen polypeptide is a kind of small molecule polypeptide prepared by fish skin as raw material through protease hydrolysis processand it has a variety of physiological function.In thi paper,Bacillus Subtilis Alkaline Protease(BAP)was used to hydrolyze tilapia collagen,BAP crosslinked enzyme aggregates(BAP-CLEAs),polymer membrane immobilized BAP and natural biological Eggshell membrane(ESM)immobilized BAP were preparaed separately.According to the results of enzymatic hydrolysis efficiency of three immobilized enzymes,ESM-BAP was selected for the fish skin collagen polypeptide preparation.The anti-osteoporosis fish skin collagen active polypeptide was separated by ultrafiltration membrane.Through osteogenic differentiation induced cell experiment in vitro and zebrafish osteoporosis activity experiment,the anti-osteoporosis fish skin collagen active polypeptide with the optimal molecular weight range was screened..The main research contents and results of this paper are as follows:(1)Study on the preparation of BAP-CLEAs by carrier free self-crosslinking method.BAP-CLEAs were successfully prepared by using the crosslinking agent genipin.When the crosslinking temperature was 35 ?,the crosslinking agent concentration was 0.50% and the crosslinking time was 12 h,the maximum enzyme activity recovery of BAP-CLEAs was 55.04%.Results of SEM,FTIR and enzymatic properties proved that genipin successfully cross-links BAP to form BAP-CLEAs.The optimum catalytic temperature of free enzyme and BAP-CLEAs was both 45 ?.The optimum catalytic p H of BAP-CLEAs was 1.0 higher than that of free enzyme,its p H stability and thermal stability were higher than that of free enzyme.However,the affinity of BAP-CLEAs to substrate was decreased.The stability of repeated use was increased,and the relative enzyme activity of BAP-CLEAs was 86.42% after 5 times of repeated use.(2)Study on immobilization of BAP on polymer membrane carrier.Cellulose acetate(CA)membrane was used as a carrierfor immobilized enzymespreparation.The maximum enzyme activity recovery of CA membrane immobilized enzyme(CA-BAP)was 57.08% under 2.5% of glutaraldehyde concentration,35 ? of crosslinking temperature,8 mg/m L of enzyme concentration and 2 h of crosslinking time,.Results of SEM,FTIR and enzymatic properties proved that enzyme molecules were successfully immobilized on the CA membrane to form the CA-BAP.Its optimum catalytic p H shifted to alkaline,the optimum catalytic temperature increased to 55 ?,and the p H stability and thermal stability were better than that of the free enzyme.Enzyme kinetics results showed that the affinity of CA-BAP to substrate is reduced and the stability of repeated use is improved.The relative enzyme activity of CA-BAP was 93.20% after 5 times of repeated use.(3)Study on immobilization of BAP on Eggshell membrane(ESM)carrier.ESM was used as a carrier and genipin was used as a cross-linking agent for immobilized enzymes praparation.The maximum enzyme activity recovery of ESM immobilized enzyme(ESM-BAP)was 64.87% when the concentration of genipin was 0.75%,the crosslinking temperature was 45 ?,the enzyme concentration was 5 mg/m L and the crosslinking time was 2 h.Results of SEM and FTIR proved that BAP was successfully immobilized on ESM.The enzymatic propertiesof ESM-BAP showed that the optimum catalytic p H of ESM-BAP was the same as that of free enzyme,the optimum catalytic temperature rose to 55? and the p H stability and thermal stability were better than that of free enzyme.The affinity of ESM-BAP to substrate and catalytic efficiency did not change much when compared with free enzyme,but the enzyme reusability was improved,and the relative enzyme activity of ESM-BAP remained 89.14% after 5 times of repeated use.(4)Preparation and activity study of anti-osteoporosis fish skin collagen active polypeptides.Comparing the enzymatic hydrolysis efficiency of BAP-CLEAs,CABAP and ESM-BAP,results showed that ESM-BAP had the highest enzymatic hydrolysis efficiency and it was applied to the preparation of anti-osteoporosis fish skin collagen active polypeptides.Through ultrafiltration of the hydrolysate,tilapia skin collagen polypeptides with molecular weights ranging from 3 k Da to 5 k Da(P5),1 k Da to 3 k Da(P3)and <1 k Da(P1)were obtained respectively.Osteoblast differentiation induction experiments confirmed that fish skin collagen polypeptides had anti-osteoporosis activity,and P1 and P3 had better effect.Accordingly,P1 and P3 were selected for the subsequent zebrafish in vivo experiment.Results proved that both P1 and P3 samples had anti-osteoporosis activity,and the anti-osteoporosis activity of P1 samples was better than that of P3 samples.