[Isolation and characterization of a cellobiose dehydrogenase formed by a asporogenic mycelial fungus INBI 2-26(-)].
A nonsporulating fungus isolated from dioxine-containing tropical soils forms cellobiose dehydrogenase, when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M(r) of 95 kDa; its pH optimum was in the range 5.5-7.0; more than 50% activity was retained at pH 4.0-8.0 (citrate-phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K(M) values at pH 6.0 equaled 4.5 +/- 1.5 and 56 microM) in the presence of dichlorophenolindophenol (K(M) app = 15 +/- 3 microM at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl-beta-D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited affinity to amorphous cellulose. At 55 degrees C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the single-electron acceptor cytochrome c3+ (K(M) app = 15 microM at pH 6.0).
Karapetian
K N
KN
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia.
Iachkova
S N
SN
Vasil'chenko
L G
LG
Borzykh
M N
MN
Rabinovich
M L
ML
D015086:2,6-Dichloroindophenol / Q000737:chemistry; D000180:Acrylic Resins; D002237:Carbohydrate Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose; D002498:Centrifugation; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned; D005658:Fungi / Q000254:growth & development / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D042844:L-Lactate Dehydrogenase (Cytochrome); D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D025282:Mycelium / Q000378:metabolism; D010084:Oxidation-Reduction; D019810:Sodium Azide / Q000737:chemistry
D000180:Acrylic Resins; D017077:Culture Media, Conditioned; D002475:Cellobiose; D002482:Cellulose; D019810:Sodium Azide; D015086:2,6-Dichloroindophenol; D002237:Carbohydrate Dehydrogenases; D042844:L-Lactate Dehydrogenase (Cytochrome); C019859:cellobiose-quinone oxidoreductase; D007785:Lactose