马尔尼菲青霉酵母细胞黑素的分离纯化及理化性质的分析

Isolation, purification and physicochemical characterization of melanin from yeast cells of Penicillium marneffei

目的探讨提取、纯化马尔尼菲青霉（PM）酵母细胞的黑素并研究其理化性质。方法在基础液体培养基中加入左旋多巴37℃振荡培养PM酵母细胞，通过溶壁酶、变性剂和浓酸等处理PM酵母细胞以分离提取黑素，用化学分析、紫外光谱、红外光谱以及电子顺磁共振（EPR）波谱研究PM黑素的理化性质。结果PM黑素与合成多巴黑素的理化性质相似，均溶解于碱，不溶于酸，不溶于水和大多数有机溶剂，在pH≤3时产生沉淀，能被氧化剂H2O2漂白。紫外光谱分析显示，在205Nm处有最大吸收峰，随波长的增加其吸收值减小。红外光谱分析表明，PM黑素具有经典的黑素红外吸收特征，即在3μm附近和6μm附近各有一个吸收峰；EPR提示PM黑素含有稳定的自由基。结论PM酵母细胞能利用外源性左旋多巴合成多巴黑素。

Objective To isolate and purify melanin from yeast cells of Penicillium mameffei （PM） and to analyze its physicochemical features. Methods PM yeast cells were cultured in minimal medium containing levodopa （L-dopa） with continuous shaking at 37 ℃. Melanin was isolated from the yeast cells of PM by cell wall- lysing enzyme, denaturant, concentrated hot acid, and purified by sodium hydroxide and concentrated hydrochloric acid. The physicochemical properties of isolated melanin were assessed on the basis of combined chemical analysis and spectroscopic methods including ultraviolet-visible （UV-vis）, Fourier transform infrared （Fr-IR） and electron- paramagnetic resonance （EPR）. Results Similar to synthetic dopa-melanin, the isolated melanin from PM was alkali soluble, acid-resistant, insoluble in water and most organic solvents, precipitated at or below PH 3, and could be bleached by hydrogen peroxide. UV-vis spectrophotometric analysis showed that the PM-derived melanin had a typical absorption peak at 205 nm and the absorbance decreased with the increase of wavelength. FT-IR spectroscopy displayed two absorption peaks at about 3 μm and 6 μm, which was characteristic of classic melanin. EPR spectroscopy revealed that the isolated melanin contained stable free radicals. Conclusion Yeast ceils of PM can use exogenous L-dopa to synthesize dopa-melanin.