【作者】 王玉霞；

【导师】 石艳；

【作者基本信息】 厦门大学 ， 生物学， 2019， 硕士

【摘要】 酪氨酸酶(TYR)(EC 1.14.18.1)是存在于人、动植物以及微生物生命活动过程中的含铜酶,它的主要功能是催化黑色素的生成。通常在紫外线的照射下,动物体内的TYR会催化黑色素的生成以抵御皮肤受损,但是皮肤色素的异常积累可能会导致严重的色素沉着障碍。同时,TYR也参与了果蔬褐变和昆虫生长发育的调控。因此,通过抑制生物体内的TYR活性来调控黑色素的产生和积累已成为当前研究的热点。胍基化合物(GCs)目前在化学合成、医疗、抗菌和生态等领域具有重要作用。本论文选取胍基硫脲(GT)、硫氰酸胍(GTC)、苯基胍(PG)、苯乙双胍(PTDG)四种GCs,运用紫外分光光度法,首先研究其对蘑菇酪氨酸酶(MT)的作用效果。从结果可以看出,四种GCs均可抑制MT单酚酶和二酚酶的活性。其中,GT、GTC、PG、PTDG抑制MT单酚酶的IC50值分别为0.27、3.53、6.68、5.18 mmol/L,抑制MT二酚酶的IC50值分别为0.23、3.24、3.97、2.50 mmol/L。其次,本论文研究了四种GCs对MT的抑制作用机理,结果表明,四种GCs对二酚酶的抑制均属于可逆抑制,说明它们的存在仅降低了酶促反应速率,而并不影响反应进程。其中,GT和PTDG是MT的竞争型抑制剂,而GTC和PG是混合型抑制剂。本论文进一步采用紫外可见光谱、内源和外源荧光淬灭法、Native-PAGE和MOE分子对接技术研究了四种GCs对MT的作用机制。MOE结果显示四种GCs均能与TYR活性中心附近的氨基酸残基相互作用,其中,GT和PTDG还可直接与活性中心处的一个铜离子结合。除此之外,本论文研究了GT和GTC对B16F10细胞和斑马鱼胚胎的黑色素生成的调节作用,结果显示,两种化合物均可抑制细胞和斑马鱼的黑色素生成量,且均下调了其TYR、小眼畸形转录因子(MITF)、多巴色素互变异构酶(DCT)的表达量。最后,本论文通过FRAP、ABTS、DPPH法和DNA氧化损伤保护法研究了四种GCs的抗氧化活性,分析显示,四种GCs中只有GT具有显著的抗氧化活性。基于以上的结果,对四种GCs对鲜切山药的保鲜效果进行了研究,结果与抗TYR和抗氧化效果一致,GT表现出较好的保鲜效果。综上所述,本论文的研究为GCs的研发提供了新的思路,四种GCs是新型小分子TYR抑制剂的潜在候选,其中,GT具有更广阔的应用前景。更多还原

【Abstract】 Tyrosinase(TYR)(EC 1.14.18.1),a copper-containing enzyme in the biological events,is usually found in human,animals,plants and microorganisms.TYR can catalyze the synthesis of melanin.For human and animals,in response to the UV light,the organism produces more melanin pigment that darkens the skin,but abnormal accumulations of skin pigmentation may cause serious pigmentation disorders.TYR is also the key enzyme in fruits and vegetables enzymatic browning and the growth and development of insects.Taken these together,the regulation of melanin generation and accumulation by the inhibition of TYR is becoming a hot spot in current research.Guanidine compounds(GCs)play an important role in fields of chemical synthesis,medicine,antibacterial and ecology.In this study,the inhibitory effects on mushroom tyrosinase(MT)of four GCs,including guanylthiourea(GT),guanidine thiocyanate(GTC),phenylguanidine(PG),and phenethyldiguanide(PTDG)were investigated.From the results,four GCs reduced monophenolase and diphenolase activities of MT.The IC50 values of GT,GTC,PG,and PTDG were 0.27,3.53,6.68,and 5.18 mmol/L for monophenolase,respectively,and were 0.23,3.24,3.97 and 2.50 mmol/L for diphenolase,respectively.The inhibitions of diphenolase activity were reversible for the four GCs.The inhibition types of GT and PTDG were competitive,while the inhibition types of GTC and PG were mixed.We further studied the mechanism of four GCs on MT by UV spectroscopy,fluorescence quenching analysis,Native-PAGE electrophoresis and molecular docking simulation as well.The results implied that four GCs could interact with the amino acid residues near the active center of TYR,GT and PTDG could form metal interactions with the copper ions of TYRBased on the inhibitory mechanism of GCs on MT,furthermore,we demonstrated the melanogenesis regulatory effect of GT and GTC on B16F10 cells and zebrafish,the results showed that the two compounds had significant anti-melanogenic activity by down-regulation of the expression of microphtalmia-associated transcription factor(MITF),tyrosinase(TYR)and dopachrome-tautomerase(DCT).The antioxidant activities of four GCs were determined by FRAP,ABTS,DPPH and DNA damage protection assays.Results revealed that GT possessed potent antioxidant activity.Based on this result,the effect of four GCs on the fresh-cut iron stick yam preservation was also presented.The results suggested that it had great potential for GCs applied to enhance freshness and quality in fruits and vegetables,and the treatment with GT had preferable storage effects.Therefore,our study provided a new idea for the research and development of GCs,the results suggested that these GCs are potential candidate of novel small molecule TYR inhibitors and GT could have broader application prospects.更多还原