Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile

Abstract

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and I1 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 11, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDSjPAGE clearly showed that the larger enzyme I1 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.Since the discovery of different cellobiose-oxidizing enzymes in cellulolytic cultures of the white-rot fungus Sporotrichum pulverulentum [l -41, similar enzymes have also been reported to occur in a variety of other cellulose-degrading fungi not taxonomically related to the former, e.g. Sporotrichum thermophile [5], Monilia sp. [6], Chaetomium cellulolyticum [7], Sclerotium rolfsii [S].The cellobiose-oxidizing enzymes of Sporotrichum pulverulentum have been extensively characterized and are of two kinds: one is a flavocytochrome (b type) having a definite oxidase activity, the other one is a simple flavoprotein. Both react with oxygen producing some superoxide radicals, but preferentially use water-soluble quinones or phenoxy radicals resulting from lignin degradation, as natural electron acceptors [2][3][4][9][10][11][12].S. pulverulentum is the anamorph stage of the basidiomycete Phanerochaete chrysosporium which, in recent years has been widely studied for its ability to degrade all wood components and particularly lignin [ 131. Indeed, the cellobiose-quinone dehydrogenase of this organism has been supposed to be functionally linked to lignin degradation. Sporotrichum (Chrysosporium, Myceliophthora) thermophile, however, is the anamorph of the ascomycete Thielavia heterothallica (Klopotek) which is a cellulolytic but not a

Genomics Review of Holocellulose Deconstruction by Aspergilli

“…Oxidation of cellobiose takes place in the flavin domain following electron transfer to the heme domain, and the reduced heme is able to reduce a wide variety of substrates, including quinones, metal ions, and organic dyes. Reduced cellobiose dehydrogenase can also react with molecular oxygen and interact with the newly discovered copper-dependent lytic polysaccharide monooxygenases that directly oxidize crystalline cellulose (15,137). The current hypothesis for the function of cellobiose dehydrogenase involves the generation of hydroxyl radicals, formed via reduction of an extracellular ferric complex (138,139) that takes part in Fenton chemistry, with hydrogen peroxide produced by CDH transferred to an array of acceptor oxidases, such as LPMOs and others that remain unknown (128,140).…”

Purification and Characterization of Cellobiose Dehydrogenase from the Plant PathogenSclerotium(Athelia)rolfsii

“…Partial proteolysis of CDH was performed as previously reported (11,52). CDH was incubated with papain (Sigma) for about 2 h, and the cyt c activity was monitored.…”

“…Alternatively, CDH activity was specifically determined by following at 550 nm the reduction of 20 M cyt c in 50 mM Na-succinate buffer, pH 3.5, containing 30 mM lactose. The extinction coefficient was 19.6 mM Ϫ1 ⅐ cm Ϫ1 (11). The pH dependence of both CDH and CBQ activity when using the indicated electron acceptors was determined using the following buffers: 50 mM malic acid (pH 1.7 to 3.0), 50 mM succinic acid (pH 2.8 to 6.4), 50 mM Tris (pH 6.9 to 8.3) for DCIP and cyt c; 50 mM acetate-50 mM morpholineethanesulfonic acid-50 mM Tris (1:1:1) (pH 2.5 to 8.0) for p-benzoquinone and DCIP; and 50 mM Na-acetate (pH 3.0 to 6.0), 50 mM Na-citrate (pH 2.0 to 6.0), 20 mM Tris buffer (pH 6.0 to 9.0) for all others.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on an Amersham-Pharmacia Multiphor II system using precast ExcelGel SDS gradient gels (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Native PAGE was carried out on an Amersham-Pharmacia Phast System using precast PhastGel gradient gels (8-25) with both high-and low-molecular-weight standards (Amersham-Pharmacia).…”

Catalytic Properties and Classification of Cellobiose Dehydrogenases from Ascomycetes

“…CDH activity was reported in cultures of the ascomycetes Thielavia heterothallica (synonyms, Myceliophthora thermophila and Sporotrichum thermophile) (3,4), Chaetomium sp. INBI 2-26(Ϫ) (38), Chaetomium cellulolyticum (10), Neurospora crassa (11), Humicola insolens (32), and Myriococcum thermophilum (14).…”