Preparation And Activity Of Tyrosinase-inhibitory Peptide From Yeast

Tyrosinase is a copper-containing enzyme that is the key rate-limiting enzyme in thesynthesis of melanin, and the synthesis of melanin in the melanocytes will be impeded whentyrosinase is inhibited by tyrosinase inhibitors. Recently a lot of synthetical tyrosinaseinhibitors which can effectly impede the synthesis of melanin have been made, however,many potent tyrosinase inhibitors have high toxicity towards cells and low stability, thuslimiting their application. Thus it was obvious to develop a new kind of tyrosinase inhibitorfrom natural sources with excellent bioactivity, safe and non-toxic. Tyrosinase inhibitorypeptide is a new tyrosinase inhibitor from natural sources, which has received much attentionin food and cosmetics fields. The yeast is a topical single microorganism, and the proteincontent in the yeast is high than50%, moreover, yeast protein is a novel wild sources, withcomplete amino acides composition and cheap. Therefore, yeast protein has high researchvalue. In this paper, yeast protein was used as a raw material, and mainly studied thepreparation, tyrosinase inhibitory activity, isolation and purification of yeast proteinenzymatic hydrolysates, their effect on the synthesis of melanin in cultured B16murinemelanoma cells.Finally, studied the mechanism of yeast active peptide was studied.1. Firstly, the paper optimized the enzymatic processing condition to produce thetyrosinase inhibitory peptides from yeast protein hydrolysates using mono-factorial andorthogonal experiment. By mono-factorial and orthogonal experiment, the optimum enzymehydrolysis conditions of papain were achieved as: pH6.5, enzyme quantity6000U/g,temperature50℃, substrate concentration1:20(w/v), hydrolysis time2h. Furthermore, YPprepared in this condition had a certain tyrosinase inhibitory activity, with the tyrosinaseinhibitory activity of42.7%(5mg/mL), and the yield of polypeptide is high up to28.85%.2. Because of the existence of polysaccharides in the enzymatic hydrolysates, the ethanolprecipitation method was used to remove these polysaccharides, and the removal rate ofpolysaccharides was45.56%for ethanol and hydrolysates volume ratio of1:1. After isolationby ultrafiltration, DEAE-52anion exchange chromatography and Sephadex G-15gel filtrationchromatography, the fraction with higher tyrosinase inhibitory activity d-1was collected. Thethe IC50of d-1was3.53mg/mL, and the molecular weight distribution of the fraction d-1wasranged from200to800Da. The fraction d-1was further purified using Sephadex G-10gelfiltration chromatography and RP-HPLC, to obtain the fraction d-1-a-2. The identification onMALDI-TOF-MS showed that the molecular weight and the sequence of d-1-a-2might be542.3Da, Phe-Phe-Asp-Asp respectively. 3. By using B16murine melanoma cell as cell model during the experiment, the effect ofyeast peptide (d-1) on the biosynthesis of melanin was studied. The result revealed that theeffect of fraction d-1on cell growth was not significant, which indicated yeast peptide had nocytotoxicity; d-1fraction decreased the biosynthesis of melanin and suppressed intracellartyrosinase activity with increasing d-1concentration. After incubation with d-1at differentconcentration (3mg/mL、4mg/mL and5mg/m) for72h, the melanin content was86.69%、74.44%and65.19%, and the intracellar tyrosinase activity was82.96%、72.11%and63.53%respectively.4. The fraction d-1showed high reducing power and lipid peroxidation inhibition activity,and had DPPH radical-scavenging activity (IC503.5mg/mL), superoxide anion-scavengingactivity (IC501.52mg/mL), and showed the hydroxyl radical-scavenging activity of37.65%atconcentration of8mg/mL; the copper-chelating ability of d-1is outstanding, and the the IC50of d-1was0.2mg/mL. Concerning the action mechanisms of d-1, blocking of the oxidativepathway and copper-chelating of enzyme activity sites may be mainly involved in inhibitingthe catalytic reaction of tyrosinase.