Investigation on Function and Molecular Structure of Cellobiose Dehydrogenase

用柱层析法从纤维分解真菌黄孢原毛平革菌和腐霉中分离纯化了两种不同的pH依赖性纤维二糖脱氢酶。纯化的酶用木瓜酶处理，分离出黄素区和血红素区。在此基础上，研究了不同pH和底物浓度条件下黄孢霉胞外黄色素纤维二糖脱氢酶(CDH)跨区电子转移的稳态前动力学。在各假体基团等色点的监测表明，黄素和血红素的还原半衰期分别为双相和单相。在所有底物浓度和所测试的pH值下，黄素还原的第二阶段的行为与血红素还原的行为几乎相同，这表明黄素半喹酮的形成和血红素还原涉及相同的电子转移反应。在pH 3.0~7.0范围内，纤维二糖对黄素有还原作用，但下一步电子转移速度随着pH的升高而降低，在pH 6.0以上几乎为零。黄素还原的第二阶段和血红素的还原同样受到高浓度底物的抑制，而黄素还原的第一阶段与纤维二糖浓度呈双曲线关系。PH的升高增强了底物对血红素还原的抑制作用，但不能增强黄素还原的底物抑制作用。白腐菌黄孢原毛平革菌的纤维二糖脱氢酶(CDH)已在甲醇营养酵母Pasforis中异源表达。表达水平达到79 mg/L，显著高于野生型CDH(WtCDH)和重组CDH(RCDH)。RCDH和wtCDH的催化作用动力学参数基本相同。

Two different pH-dependent cellobiose dehydrogenases were purified from cellulolytic fungi Phanerochaete chrysosporium and Humicola insolens by column chromatographies. The purified enzymes were treated with papain to separate into flavin and heme domains. Redox potentials of both prosthetic groups were determined.Furthermore, the pre-steady-state kinetics of inter-domain electron transfer in the extracellular flavocytochrome cellobiose dehydrogenase (CDH) from P. chrysosporium was studied under various conditions of pH and substrate concentration. Monitoring at the isosbestic point of each prosthetic group indicated that the reductive half-reactions of flavin and heme were biphasic and monophasic, respectively. The behavior of the second phase of the flavin reduction was almost identical to that of heme reduction at all substrate concentrations and pH values tested, suggesting that the formation of flavin semiquinone and heme reduction involve the same electron transfer reaction. Although flavin reduction by cellobiose was observed in the range of pH 3.0 to 7.0, the velocity of the next electron transfer step decreased with increase of pH and was almost zero above pH 6.0. The second phase of flavin reduction and the heme reduction were inhibited similarly by high concentrations of substrate, while the first phase of flavin reduction showed a hyperbolic relation to the cellobiose concentration. Increase of pH enhanced the substrate inhibition of heme reduction but not the initial flavin reduction.Cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pasforis. The expression level reached 79 mg/L of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. The kinetic parameters of catalytic function for rCDH were nearly identical to those of wtCDH.