Study On Bioactive Peptide From Laver Hvdrolvzed By Protease

Development and utilization of marine resources with rich varieties is one of the hottopic of people around the world. Bioactive peptides coming from marine protein resourcesby controlled enzymolysis have broad application prospects in the fields of medicine, food,feed, cosmetics and health care products. In this paper, the laver coming from Jiangsu coastwas used as a raw material to prepare laver protein antimicrobial peptides (LPAP) and laverα-glucosidase inhibitor (LGI) by enzymolysis under different conditions. In addition, theseactive components were purified and separated. Meanwhile their composition andcharacteristics were studied respectively.Neutral protease, papain, acid protease, alkaline protease, pepsin, and trypsin were usedto hydrolyze laver protein under optimum conditions and papain was chosen as thehydrolyzing enzyme by protein hydrolysate against indicator (Staphylococcus aureus). Thehydrolysis conditions were optimized as follows: substrate concentration5%,1.0×104U g-1papain, pH6.5, temperature65oC, and hydrolysis time4h.The relatively molecular weight distribution of laver enzymolysis liquid showed thatlaver protein antibacterial peptides with molecular weight below1000Da accounted for96.68%. The LPAP was purified through DEAE-52anion exchange chromatography andSephadex G-10gel filtration chromatography, which was identified as a tetrapeptide FFDD(Phe-Phe-Asp-Asp) by Q-TOF-MS. This LPAP possessed a relatively broad spectrum ofantibacterial activity, and showed signifcant growth inhibition against G+bacterials and G-bacterials. Minimum inhibitory concentration of LPAP against indicator was0.25mg mL-1atthe optimal pH6.5. The result showed that2mg mL-1antibacterial peptides had the sameinhibitory effect with36μg mL-1ampicillin or100μg mL-1kanamycin against S. aureus. Theelectron microscope photos of the indicator bacteria impacted by antibacterial peptidesrevealed its antibacterial mechanism is mainly due to the destruction of the cell membrane.Combined proteolyzation of laver by neutral protease, alkaline protease andaminopeptidase was optimized using the α-glucosidase activity inhibitory rate as index.Hydrolysate mainly contains small molecular peptide, and those with molecular weight below1500Da accounted for82.47%.LGI was decolorated by actived carbon, and further purified through SP Sepharose HighPerformance cation-exchange chromatography, Sephadex G-10gel filtration chromatographyand Mono Q anion exchange chromatography. The IC50of LGI against α-glucosidase was97.36μg mL-1, with the molecular weight of231.2Da and the amino acid sequence wasidentified as Ala-Ala-Ala by Q-TOF-MS. The kinetic parameters of reaction betweenα-glucosidase and LGI indicated that LGI was one of the non-competitive inhibitors. By thecircular dichroism, the change of α-glucosidase’s structure interacted with LGI showed thevalue of α/β of α-glucosidase increased by6.40times from2.15to13.97, leading to anobvious change in α-glucosidase conformation compared to the native state, and activitydecreased significantly. LPAP and LGI obtained from marine natural material by enzymolysis were peptides withlower molecular weight which are safe and easy to be absorbed. Meanwhile their stability totemperature and pH provided the basis for comprehensive utilization of laver.