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

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An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C. 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro. Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides. Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced. Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+. The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors. As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.

A strain of Sporotrichum thermophile (var. 2), grown in submerged culture at 44 "C, decayed crystalline cellulose by the secretion of cellulolytic enzymes into the culture medium.Dialysed culture filtrates, after growth on cellobiose, wese also effective in degrading crystalline cellulose (Avicel). Cellulase, measured viscometrically with carboxymethylcellulose as substrate and therefore referred to as C M-cellulase, was induced by cellulose, glucomannan, cellobiose and, to a limited extent, by laminaribiose and cellobionic acid. Low CM-cellulase activities were also detectable when the organism was grown on other carbon sources. In culture media supplemented with readily metabolized non-inducing substrates, such as glucose, CM-cellulase activity did not increase after their exhaustion indicating that no derepression of constitutive CM-cellulase occurred. CM-cellulase synthesis induced during growth on cellobiose was strongly inhibited, although not completely suppressed, by glucose and other carbon sources. In cultures grown on glucose and cellobiose, glucose was utilized first and CM-cellulase synthesis was repressed ; cellobiose utilization occurred only after glucose exhaustion and triggered CM-cellulase formation. When glucose was added to a culture growing on cellobiose, further utilization of the latter was prevented and CM-cellulase synthesis was inhibited. The use of glucose analogues gave some indications that cellobiose did not compete with glucose for the same transport carrier and that glucose catabolism was a prerequisite for the inhibition of CM-cellulase synthesis.

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