Abstract
Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity of P. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains of P. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3-9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain from H. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor from H. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.
Highlights
Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens
From the absorption spectra of each band, as described it was concluded that the bands corresponding to molecular masses 35 and 55 kDa were the heme and flavin domains of P. chrysosporium CDH, respectively, in accordance with the results reported by Henriksson [17]
Three CDH fractions were isolated from H. insolens crude cellulase, whereas only one CDH was contained in culture solution of P. chrysosporium
Summary
Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. Recent investigations have demonstrated that another cellobiose-oxidizing flavoprotein, cellobiose:quinone oxidoreductase (CBQ, EC 1.1.5.1) [9, 10], is the flavincontaining domain of CDH produced by proteolytic activity [11,12,13] In addition to their catalytic function, both CDH and CBQ can bind to cellulose as well as many cellulases [11, 14]. It has been reported that both cytochrome c and DCPIP were reduced by CDH at pH 4.2, whereas only DCPIP was reduced effectively at pH 5.9 This phenomenon was explained by stopped-flow kinetic study that both flavin and heme were reduced at a high rate at pH 4.2, whereas only flavin reduction proceeded quickly at pH 5.9 [18]. The reduction of cytochrome c is dependent on heme, and an electron is transferred from cellobiose to this electron acceptor via both FAD and heme, whereas the reduction of DCPIP is catalyzed only by flavin
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