[目的]筛选和鉴定三角帆蚌(Hyriopsis cumingii)珍珠层蛋白酶解产物中的酪氨酸酶抑制肽，为美白肽的研究与开发提供依据。[方法]以三角帆蚌珍珠层粉为原料，基于其蛋白组成特性，采用物理改性和可控酶解技术制备珍珠层活性肽(Peptides from nacre powder，P-NP)，通过测定其2，2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)和1，1-二苯基-2-苦基肼自由基(DPPH)的清除能力、酪氨酸酶抑制活性以及对紫外线B(UVB)辐照损伤人永生化角质形成细胞(HaCaT)的影响，探究其体外活性。采用液质联用技术(LC-MS/MS)和分子对接技术从P-NP中鉴定并筛选出三角帆蚌酪氨酸酶抑制肽，并进行固相合成和体外活性验证。[结果]P-NP的ABTS和DPPH自由基清除效果较佳，肽质量浓度为1。00、14。00 mg/mL时，两者清除率分别为41。58％和11。23％;P-NP的酪氨酸酶活性抑制能力良好，其半抑制浓度(IC50)值为7。51 mg/mL。P-NP质量浓度在83。22µg/mL时，显著提高UVB损伤HaCaT细胞的生长活力(P<0。05)。P-NP经LC-MS/MS鉴定后筛选出相对分子质量集中在500～1000的61条肽序列作为候选肽库，分子对接筛选出3条潜在的酪氨酸酶抑制肽，其主要通过氢键与酪氨酸酶活性中心及其以外的位点相结合，从而达到抑制其活性的作用。其中，Tyr-Pro-Asn-Pro-Tyr(YPNPY)对酪氨酸酶抑制作用最强，IC50为(0。545±0。028)mmol/L，主要抑制类型为竞争型抑制。[结论]三角帆蚌珍珠层活性肽具有良好的清除自由基和酪氨酸酶抑制活性。
Screening and Activity Evaluation of Tyrosinase Inhibitory Peptides from Hyriopsis cumingii Based on Molecular Docking
[Objective]To identify and screen the whitening factor tyrosinase-inhibiting peptides from enzymatic hydrolysate of freshwater pearl mussels Hyriopsis cumingii,and provide a basis for the research and development of whitening peptides.[Method]The pearl active peptide (Peptides from nacre powder,P-NP) was prepared from the nacre powder of H.cumingii (NP-Hc) by physical modification and controlled enzymatic technology,based on the protein composition of the nacre powder,and its activity in vitro was explored by measuring the scavenging capacity of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) and 1,1-diphenyl-2-picrylhydrz (DPPH),and tyrosinase inhibitory capacity,and the effect of P-NP on the ultraviolet B (UVB) irradiation damaging human immortalised keratinocytes (HaCaT).Liquid chromatography-mass spectrometry (LC-MS/MS) and molecular docking techniques were used to identify and screen H.cumingii tyrosinase inhibitory peptides from P-NP,which were solid-phase synthesized and then verified for in vitro activity.[Result]P-NP had a good scavenging effect on ABTS and DPPH free radicals (41.58％ and 11.23％ at the concentration of 1.00 mg/mL and 14.00 mg/mL,respectively),and showed good inhibition of tyrosinase activity with an IC50 value of 7.51 mg/mL.P-NP (with peptide concentration of 83.22 µg/mL) significantly increased the growth viability of UVB damaged HaCaT (P<0.05).Sixty-one peptide sequences,identified from P-NP by LC-MS/MS and with a relative molecular mass of 500-1000,were selected to establish a candidate peptide library.And three potential tyrosinase inhibitory peptides screened by molecular docking mainly bind to the active center of tyrosinase through hydrogen bond,thus achieving the inhibition activity.Among them,the peptide Tyr-Pro-Asn-Pro-Tyr (YPNPY) had the strongest inhibitory effect on tyrosinase,with an IC50 of (0.545±0.028) mg/mL and a competitive inhibition type.[Conclusion]The active peptides derived from pearl layer of H.cumingii have good free radical scavenging and tyrosinase inhibiting activities.
万方数据
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