短梗霉黑色素的分离纯化及结构的初步分析

Isolation,Purification and Structural Analysis of Melanin from Aureobasidium pullulans

采用热碱提取、水煮酸沉法从短梗霉发酵液中提取得到黑色素粗品,再经DMSO萃取、酸性甲醇(pH=2)沉淀得到不含多糖和蛋白的短梗霉黑色素。此黑色素不溶于水及常规有机溶剂,可溶于碱性溶液和DMSO;离子交换色谱分析表明黑色素组分均一,出峰时间26±0.5 m in;紫外光谱谱图最大吸收峰为215 nm左右,未见蛋白(280 nm)与核酸(260 nm)的特征吸收峰;红外光谱谱图具有黑色素3μm和6μm的特征吸收峰,并含大量的羟基、氨基,与核磁共振和液质联机谱图结合分析推出短梗霉黑色素可能含有酚羟基、羧基和吲哚等官能团,主要结构骨架为5,6-二羟基吲哚-羧酸和多巴醌,推断该黑色素为酪氨酸酶控制合成的真黑素。

Crude melanin was obtained from fermentation fluid of Aureobasidium pullulans by means of hot NaOH extraction and HC1 precipitation. Then from this crude melanin through DMSO extraction and HCl- methanol （ pH = 2） precipitation, the melanin without polysaecharide and protein could be obtained. The melanin was not dissolved by water and general organic solvent but alkaline solution and DMSO; The ion exchange chromatograph analysis indicated the melanin component was homogeneous, the peak with retention time 26 ±0.5 min ; Ultra -violet spectrogram indicated that the absorption peak was about 215nm, but the characteristic absorption peaks of protein （280nm） and nucleic acid （260nm） had not been seen; The infrared spectrogram indicated that the melanin had the characteristic absorption peaks of 3 μm and 6 μm, abound in hydroxyl and amino; in addition, from the analysis combining the nuclear magnetic resonance with high performance liquid chromatography coupled with mass spectrometry （ HPLC - MS）, it could be inferred that the melanin possibly contained amido, hydroxyl, methyl - oxygen and carboxyl functions with its main structural framework of 5,6 - 2 - hydroxy indole - carboxylic acid and dopaquinone. The melanin belongs to the eumelanin in the control of tyrosinase.