Shaw C E, Kapica L
Appl Microbiol. 1972 Nov;24(5):824-30. doi: 10.1128/am.24.5.824-830.1972.
Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall.
新型隐球菌在由土豆和胡萝卜提取物、蚕豆（蚕豆属）或埃塞俄比亚画眉草种子制成的琼脂培养基上生长时会产生棕色色素菌落。由于其他酵母不产生这种色素，这些培养基可用作新型隐球菌的鉴别分离培养基。新型隐球菌在含有六种不同酚氧化酶底物（邻二酚：氧氧化还原酶EC 1.10.3.1）的化学限定琼脂培养基上也产生了类似的特异性色素，该酶催化邻二酚氧化为黑色素。底物单独添加到培养基中，包括L-3,4-二羟基苯丙氨酸（L-DOPA）、绿原酸、原儿茶酸、儿茶酚、去甲肾上腺素和3-羟基酪胺盐酸盐（多巴胺）。没有底物的培养基上不产生色素。通过测量L-DOPA溶液在480 nm处吸光度的增加来测定新型隐球菌无细胞提取物的硫酸铵沉淀物中的酚氧化酶活性。该反应显示有氧气摄取，并且被铜螯合剂有效抑制，但不被过氧化氢酶抑制。该酶还氧化了其他五种诱导色素形成的底物。在L-DOPA中孵育的细胞的电子显微镜照片显示色素沉积在细胞壁中。
