| Haliotis discus hannai is the main breed of abalone in Shandong Peninsula,but about30-40%of it’s soft tissue organs are usually discarded as scraps during processing.There are a variety of active biological enzymes in abalone organs.In order to be more effective to develop and utilize the organs of abalone to reduce environmental pollution,in this paper,from the digestive tract and digestive glands of Haliotis discus hannai,digestive enzymes with high activity were obtained and the extraction conditions of the enzymes were optimized accordingly.After that,the obtained abalone digestive enzymes were preliminarily screened,and the screened enzymes were separated and purified,and their enzymatic reactions were studied.Finally,comparing the degradation effects of commercial enzymes purchased on the market and abalone digestive enzymes on Asparagus,the conclusions are as follows:1)The extraction results of abalone digestive enzymes showed that three enzymes with higher activity were amylase,cellulase,and algin lyase.The optimal extraction conditions of cellulase was 50%～60%of ammonium sulfate,the optimal concentration of PBS extraction buffer was 0.10 mol/L,p H was 5.5,and the content of cellulase extracted under this condition was 73.8 mg;the optimal extraction condition of amylase was 40%～50%of ammonium sulfate,the optimal concentration of PBS extraction buffer was 0.10 mol/L,the p H was 6.0,and the content of amylase extracted under this condition was 115.5 mg;the alginate lyase ammonium sulfate was salted out in stages,the optimal addition amount of ammonium sulfate was 80%～90%,the optimal concentration of PBS extraction buffer was0.05 mol/L,the p H was 8.2,and the content of alginate lyase extracted under this condition was 14.79 mg.2)The amylase and cellulase in abalone digestive enzymes were separated and purified by the combination of ultrafiltration and CM-52 cation exchange chromatography,in which the peak tube specific activity of cellulase activity was 229.83 U/mg;After several steps of purification,the final specific activity of cellulase increased by 6.45 times,and the yield was 17.08%;in the amylase CM-52 cation exchange chromatography,and the peak enzyme activity of amylase was 509.96 U/mg;After several steps of purification,the specific activity of abalone amylase was increased by 4.95 times,and the yield was 19.01%.3)The results of cellulase enzymatic kinetics showed that when 0.44 mg of cellulase was added,the substrate was excessive when the substrate concentration was 8 mg/m L,and the optimum p H for the enzymatic reaction was 6.0,The optimum temperature was 45℃,and the enzyme reaction time is suitable to be less than 30 min when determining the enzyme reaction rate of cellulose;the Km value of cellulase was 1.7643 mg/m L,and the Vmax was1.8975 mg·L-1·min-1.4)The results of amylase enzymatic kinetics showed that when 0.22 mg of amylase was added,the substrate was excessive when the substrate concentration was 3 mg/m L,the optimum p H of the substrate was 6.0,and the optimum temperature of the enzymatic starch solution was 40°C,when measuring the enzymatic reaction rate of amylase,the reaction time should be within 40 min;the Km value of amylase was 1.4734 mg/m L,and the Vmax was 3.8329 mg·L-1·min-1.5)The technology of the digestive enzyme of abalone to hydrolyze the mustard greens was studied,and compared it with the commercial enzyme.The orthogonal test of the commercial enzyme found that the enzymatic hydrolysis temperature had the strongest effect on the enzymatic hydrolysis of Gracilaria Lemaneiformis,which main effect was the most obvious,followed by the cellulase addition>time>the amylase addition>p H,and the material to liquid ratio has the least influence;the final optimal enzymatic hydrolysis conditions are cellulase addition of 1.2%,amylase addition of 1.2%,enzymatic hydrolysis time 2.5 h,enzymatic hydrolysis temperature 50℃,p H 7.0,material to liquid ratio 1:25;the orthogonal test of abalone digestive enzymes found that the cellulase addition had the strongest influence on the enzymatic hydrolysis reaction of Gracilaria Lemaneiformis,which main effect was the most obvious,followed by amylase addition>temperature>solid-liquid ratio>p H,and the effect of enzymatic hydrolysis time is the lowest;the optimal conditions are cellulase addition 1.6%,amylase addition 0.6%,enzymatic hydrolysis time2.5 h,enzymatic hydrolysis temperature 40℃,p H 6.0,solid-liquid ratio 1:15.6)After optimization by orthogonal experiment,the average degree of polymerization of Astragalus under the optimal enzymatic hydrolysis condition of commercial enzyme was4.0032,while the result of the average degree of polymerization of Astragalus under the optimal enzymatic hydrolysis condition of abalone digestive enzyme was 1.9892.Obviously,the effect of abalone digestive enzyme enzymatic hydrolysis of Astragalus is better than that of commercial enzymes. |
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