Cloning and Characterization of a Thermostable Cellobiose Dehydrogenase fromSporotrichum thermophile

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. CDH contains one hemeband one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochromec.In this report, a thermostable CDH from the thermophilic ascomyceteSporotrichum thermophilehas been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60°C, and the activation energy for the reaction was 26.3 kJ/mol. TheKmandkcatwere temperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library ofS. thermophilewith a polyclonal antisera raised againstPhanerochaete chrysosporiumCDH. The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da.S. thermophileCDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases fromTrichoderma reesei.Comparison with the CDH sequences ofP. chrysosporiumandTrametes versicoloridentified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs. With regard to the amino acid composition,S. thermophileCDH has more disulfide linkages and acidic and basic amino acids compared to CDHs fromP. chrysosporiumandT. versicolor.