Karapetian K N, Iachkova S N, Vasil'chenko L G, Borzykh M N, Rabinovich M L
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia.
Prikl Biokhim Mikrobiol. 2003 Nov-Dec;39(6):642-51.
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).
从含二恶英的热带土壤中分离出的一种非产孢真菌，在添加纤维素源的培养基中生长时会形成纤维二糖脱氢酶。通过SDS-PAGE纯化至同质的该酶（产率43%）的分子量为95 kDa；其最适pH值在5.5 - 7.0范围内；在pH 4.0 - 8.0（柠檬酸盐-磷酸盐缓冲液）下保留超过50%的活性。该酶在可见光范围内的吸收光谱具有黄素细胞色素蛋白的特征外观。在以二氯酚靛酚（pH 6.0时的表观K(M) = 15 ± 3 μM）作为电子受体存在的情况下，纤维二糖脱氢酶氧化纤维二糖和乳糖（pH 6.0时各自的K(M)值分别等于4.5 ± 1.5和56 μM）。其他糖类即使被该酶氧化，程度也极低。即使在100倍摩尔过量的条件下，乙基-β-D-纤维二糖苷、庚糖二糖和壳三糖都不会抑制乳糖的酶促氧化。该酶被叠氮化钠对二氯酚靛酚还原的反应微弱抑制，并对无定形纤维素表现出亲和力。在55℃和pH 6.0（最佳稳定性）下，达到最大失活一半的时间为99分钟。被纤维二糖还原的酶比未还原形式更稳定。相反，在pH 6.0时，氧化剂（二氯酚靛酚）的存在使稳定性降低了八倍。此外，该酶作为单电子受体细胞色素c3+的有效还原剂（pH 6.0时的表观K(M) = 15 μM）。
