Schmidhalter D R, Canevascini G
Institut de Biologie végétale et de Phytochimie, Université de Fribourg, Switzerland.
Arch Biochem Biophys. 1993 Feb 1;300(2):559-63. doi: 10.1006/abbi.1993.1077.
The cellobiose dehydrogenase secreted by Coniophora puteana (Schum ex Fr) Karsten during growth on cellulose was isolated by successive anion-exchange chromatography on Q Sepharose fast flow and on TSK DEAE-650S and gel filtration on Superose 12. The enzyme was recovered at a 41% yield with a 43-fold increase in specific activity. The purified sample was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium lauryl sulfate (SDS)-PAGE, and electrophoretic titration curve analysis and stained positively for glycoprotein (periodic acid/Schiff base reaction) and hemoprotein (peroxidase reaction). By isoelectric focusing over a narrow pH range two distinct bands were observed: a major band (pI 3.9) flanked by a minor band on its acidic side. FPLC gel filtration on TSK G3000 SW revealed a M(r) of 192,000, whereas on SDS-PAGE a single band, corresponding to a M(r) of 111,000, was observed. The enzyme contained 13% sugar as mannose and upon digestion with endoglycosidase H, its molecular weight was lowered by 11 kDa. The enzyme showed a visible spectrum compatible with that of a b-type cytochrome containing a flavin cofactor. It was able to oxidize cellobiose, cellodextrins, and lactose at their C1-reducing group, with dichlorophenol indophenol as oxidant. Oxygen consumption (oxidase reaction in a Clark electrode) was not at a detectable rate. Km and Vmax for cellobiose oxidation were 84 microM and 2.98 mumol mg-1 min-1, respectively, but the enzyme was strongly substrate (cellobiose) inhibited (Kis 5.4 mM).
在纤维素上生长期间，由卧孔菌（Coniophora puteana (Schum ex Fr) Karsten）分泌的纤维二糖脱氢酶，通过在Q Sepharose fast flow和TSK DEAE - 650S上连续进行阴离子交换色谱以及在Superose 12上进行凝胶过滤而被分离出来。该酶的回收率为41%，比活性提高了43倍。通过聚丙烯酰胺凝胶电泳（PAGE）、十二烷基硫酸钠（SDS）- PAGE和电泳滴定曲线分析，纯化后的样品是均一的，并且对糖蛋白（过碘酸/席夫碱反应）和血红素蛋白（过氧化物酶反应）呈阳性染色。通过在狭窄的pH范围内进行等电聚焦，观察到两条不同的条带：一条主要条带（pI 3.9），在其酸性一侧有一条次要条带。在TSK G3000 SW上进行FPLC凝胶过滤显示分子量为192,000，而在SDS - PAGE上观察到一条对应分子量为111,000的单一泳带。该酶含有13%的糖，以甘露糖形式存在，用内切糖苷酶H消化后，其分子量降低了11 kDa。该酶的可见光谱与含有黄素辅因子的b型细胞色素的光谱一致。它能够在其C1还原基团处氧化纤维二糖、纤维糊精和乳糖，以二氯酚靛酚作为氧化剂。氧消耗（在克拉克电极中的氧化酶反应）速率不可检测。纤维二糖氧化的Km和Vmax分别为84 μM和2.98 μmol mg-1 min-1，但该酶受到强烈的底物（纤维二糖）抑制（Kis 5.4 mM）。
