Canevascini G, Borer P, Dreyer J L
Institut de Biologie végétale et de Phytochimie Université de Fribourg, Switzerland.
Eur J Biochem. 1991 May 23;198(1):43-52. doi: 10.1111/j.1432-1033.1991.tb15984.x.
Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.
嗜热侧孢霉（金孢子菌）ATCC 42464的两种纤维二糖脱氢酶经DEAE - Trisacryl色谱分级分离后，分别命名为纤维二糖脱氢酶I和II，通过不同的色谱技术均已纯化至同质。这两种酶均为轻度糖基化的黄素细胞色素b蛋白，具有相似的催化特性，但分子量不同（酶I和II分别为91 kDa和192 kDa），等电点也不同（分别为4.1和3.45）。SDS/PAGE检测清楚地表明，较大的酶II是一种同二聚体，其亚基与脱氢酶I接近，但不同，脱氢酶I在该技术下是同质的。用木瓜蛋白酶对这两种酶进行有限消化后，形成了两个具有残余活性的主要部分，一个携带血红素，另一个是黄素成分；每个部分都以其特定的色谱行为为特征。携带黄素的成分显示出非典型的（对于黄素蛋白）三条带光谱，表明存在黄素衍生物。这两种酶与氧气反应非常缓慢，明显形成一些超氧自由基并可能生成过氧化氢。纤维二糖和其他纤维糊精在其还原性糖基部分被氧化为相应的醛糖酸。使用可自氧化的吩嗪硫酸甲酯时，纤维素（无论是水合形式还是结晶形式）在游离还原端也会被氧化，因此在酶促水解后会释放出相当数量的纤维二糖酸。
