Properties of neutral cellobiose dehydrogenase from the ascomycete Chaetomium sp. INBI 2-26(-) and comparison with basidiomycetous cellobiose dehydrogenases.
The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.
Karapetyan
K N
KN
A.N. Bach Institute of Biochemistiy, Russian Academy of Sciences, 33, Leninsky Prospect, 119071 Moscow, Russia.
Fedorova
T V
TV
Vasil'chenko
L G
LG
Ludwig
R
R
Haltrich
D
D
Rabinovich
M L
ML
D015086:2,6-Dichloroindophenol / Q000737:chemistry; D001487:Basidiomycota / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000032:analysis* / Q000737:chemistry; D002475:Cellobiose / Q000737:chemistry; D002597:Chaetomium / Q000201:enzymology*; D003574:Cytochrome c Group / Q000737:chemistry; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000737:chemistry; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D003574:Cytochrome c Group; D005656:Fungal Proteins; D002475:Cellobiose; C017165:cytochrome c(3); D015086:2,6-Dichloroindophenol; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase; D007785:Lactose