| Nemipterus virgatus is one of main fishery products in south China sea and is main catch of dredge and fishing. In this paper techniques of enzymology, G-PAGE and molecular biology were applied to study the distribution and characteristic of digestive protease drived from Nemipterus virgatus, ,changing regularity of stomach proteases; The kinetics,spectra and mechanism of action were also investigated during the cource of alkaline denaturation,inhibitory action and activation by Cu2+. intrinsic mechanisms of enzymatic hydrolysis of plant and animal protein with pepsin was analysed.The results of distribution and properties of three groups of Nemipterus virgatus showed that the activities of protease in all digestive organs went up with body weight increasing, but there was not ratio between the activity and body weight. Not only the activity but also the specieses and amonts was different with the changing of body weight. The protease activity was in descending order, pylorus caeca>stomach> foregut>midgut>hindgut>hepatopancreas. But maximal total protelytic activity was found in stomach.The optimal extracting parameters were obtained by single factor experiment and response surface methodology. The extracting condition was extracting time as 6h, extracting temperature at 23℃, pH 5,the activiy got 1881U/g·min. Crude stomach proteases were purified by ammonium sulphate precipitate, DEAE-Sepharose Fast Flow negion exchange chromatography exchange chromatography and Sephacryl S-200 gel filtration chromatography. The specific activity of purified protease was increasing from 17.4 to 184.4U/mg, purified multiplewas up to 10.6 yield was 14.1%. Purified protease exhibit a single band on SDS-Polyacrolymide gel and the molecular weights are determined to be about 36600Dal.Maximal acid protelytic activity was found at pH 2.5. High stability in the acid and neutral ranges except for pH 4 and marked loss of activity at alkaline pH were noticed. Optimal temperature was 40℃when reacting for 10 min and shift of optimal temperature was observed when being prolonged to 90 min. Acid protelytic activity decreased with increased NaCl concentration. The result of the activity was fully inhibited by Pepstatin A and partly by 2-ME,NBS and AcBr, but not by SBTI or EDTA. The result indicate that purified stomach protease belongs to pepsin. The value of kinetic constant Km, kcat and kcat / Km of stomach protease was determined to be 0.0684mmol/L, 101S-1,1485mmol-1·S-1·L. According to spectra of FTIR, fluorescence and circular dichroism, the alkaline denature cause to change of secondary structure, but primary structure was not changenable. Effect of Pepstatin and Cu2+ on the pepsin, cause to breakage and recolonization of band, so its primary structure was changenable. The ratio ofα-helix andβ-sheet decreased and random increased due to alkaline denature and pepstatin. The ratio of turn decreased and that ofβ-sheet increased. So combinating the spectra of FTIR, fluorescence and circular dichroism at three conditions, the ratio ofβ-sheet was high and tertiary structure belonged toα/βmodle in the pepsin structure.Differential scanning calorimetry (DSC) and SDS-PAGE electrophoretic were employed to analyze the thermal properties of soybean isolate protein(SPI)and hydrolytic velocity of different subunit. In native SPI, the acidic subunits of glycinin was easier to be degraded by pepsin than basic subunits. Inβ-conglycinin the order of hydrolysis from easy to difficult wasα-subunits,α′-subunits andβ-sub-units. After heat-treating,α- ,α′- andβ-were all degreded. The results showed heat denaturation increased the release of free amino acids, low-molecular-weight peptides and the recovery of soluble nitrogen. Therefore, thermal modification could be used to produce the peptides with different molecular weight distribution and enhanced functional properties of protein hydrolysates.Difference on characteristic of food protein hydrolysate with Nemipterus virgatus and other pepsins. Myofibrillar protein-hydrolysate was same in DH;Hydrolysate of myofibrillar with pepsin of Nemipterus virgatus was higher in free amino acid and the nitrogen released ratio, lower in hydrophobicity value. Hydrolysate of myofibrillar with pepsin of Nemipterus virgatus was higher in DH, free amino acids and the nitrogen released ratio, lower in hydrophobicity value. According to the above, pepsin of Nemipterus virgatus has wide applying perspective in the sauce products.
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