Preparation,Identification And Activities Of Tyrosinase Inhibitory Peptide From Walnut Residue

Walnut(Juglans regia L.)is one of the most common nut crops on the market with rich nutritional and medicinal value.A lot of residues are produced during the walnut oil production,which are usually sold as feed at a low price.Which not only caused the waste of walnut protein resources,but also seriously hindered the development of the walnut industry.Bio-enzymatic technology as a biological means,can improve the biological utilization of substrate.On the other hand,the protein of walnut could be degraded to small molecule compounds,which are beneficial to the body's absorption and helpful to increase the functional activities and the nutritional value.In this study,walnut residue used as raw material to obtain the polypeptide by bio-enzymatic method.Hydrolyzing walnut protein with different proteases to determine the optimal hydrolyzing protease.Response surface method was used to optimize the enzymatic hydrolysis processing of walnut polypeptide.The enzymatic hydrolysate was separated and purified by ultrafiltration and gel filtration chromatography.The inhibition rate of tyrosine monophenolase and diphenolase was determined and the polypeptide component with higher tyrosinase inhibitory activity was obtained.The composition of amino acids and the molecular weight distribution of the purified walnut polypeptide was measured by LC-MS/MS.The target peptide was determined by molecular docking with Autodock Vina,and the target polypeptide was synthesized and studied for tyrosinase inhibition kinetics and vitro digestion stability.The results were as follows:Alkaline protease showed certain advantages in peptide yield and tyrosinase inhibition rate,and it was consequently selected as the optimal protease used in hydrolyzing walnut protein.The optimal condition of walnut protein hydrolysis with alkaline protease by response surface method was as follows:temperature was 61?,substrate concentration was 5.1%,pH was 10.1.Under these conditions,the actual peptide yield was 30.18±0.26%,which was close to the theoretical value.The walnut polypeptide component F1 with good tyrosinase inhibitory activity was obtained by ultrafiltration and Sephadex G-25 gel chromatography.The identification by LC-MS/MS showed that the component F1 contained 606 polypeptide fragments with the number of amino acids is 10 or less.Molecular docking results showed that FPY had the lowest docking energy,which was-8.2 kcal/mol,indicating that FPY is the most stable ligand combined with tyrosinase.Hence,the sequence of the walnut peptide with higher tyrosinase inhibitory activity was Phe-Pro-Tyr,and the molecular weight was 498.23 Da.The docking conformation of FPY with tyrosinase showed that FPY is mainly docked in the groove of the receptor protein tyrosinase(2Y9X).There are three main types of interaction between them:hydrogen bonding,hydrophobic interaction and electrostatic interaction.Results of enzyme kinetic showed that synthetic FPY could inhibit the monophenolase and diphenolase activities.Measured IC50 values of the inhibition on the tyrosine monophenolase and diphenolase of FPY,which were 0.47±0.01 mg/mL and 1.37±0.03 mg/mL,respectively.The inhibition type of tyrosinase monophenolase was reversible competitive inhibition and the michaelis constant Km was 3.56 mmol/L and the inhibition constant Ki was 22.04 mmol/L.The inhibition type of tyrosinase diphenolase was reversible competitive inhibition and the michaelis constant Km was 4.09 mmol/L and the inhibition constant Ki was 4.82 mmol/L.And kept their tyrosinase inhibitory activities under the action of vitro gastrointestinal protease.