Karapetyan K N, Fedorova T V, Vasil'chenko L G, Ludwig R, Haltrich D, Rabinovich M L
A.N. Bach Institute of Biochemistiy, Russian Academy of Sciences, 33, Leninsky Prospect, 119071 Moscow, Russia.
J Biotechnol. 2006 Jan 2;121(1):34-48. doi: 10.1016/j.jbiotec.2005.06.024. Epub 2005 Aug 19.
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.
从毛壳菌属INBI 2-26(-)中获得的胞外纤维二糖脱氢酶（CDH）分子量为95 kDa，等电点为5。这种新型CDH对纤维二糖（K(m,app) 4.5 microM）和乳糖（K(m,app) 56 microM）的氧化具有高度特异性。以2,6-二氯靛酚（DCIP）和细胞色素c(3+)（cyt c(3+)）作为电子受体时，CDH在pH 6时活性最高。在20℃和pH 6条件下，该酶对纤维二糖、乳糖、DCIP和cyt c(3+)的转换数在9-14 s(-1)范围内。紫外可见光谱揭示了该酶的黄素血红蛋白性质。该酶的细胞色素b结构域在广泛的pH范围内可被抗坏血酸、连二亚硫酸盐还原，也可被纤维二糖特异性还原。还原血红素结构域自发再氧化的表观一级速率常数在pH 4.5和6.5时分别估计为0.01和0.00039 s(-1)。CDH在pH 6和55℃下的半失活时间约为100分钟；在pH 8，尤其是pH 4时稳定性明显较低。纤维二糖可使该酶稳定，抵抗热失活，而DCIP则会使该酶敏感化。这种新酶对结晶纤维素的亲和力较低，但能够结合到磷酸膨胀滤纸。结果表明，与已知的CDH存在显著差异，并且在一些生物技术应用方面具有前景，其中在中性pH下具有最大活性且对纤维二糖和乳糖具有高亲和力的CDH可能具有一些优势。
