INRA, UMR1163 Biotechnologie des Champignons Filamenteux, Aix-Marseille Université, ESIL Polytech Marseille, 163 avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France.
Appl Microbiol Biotechnol. 2013 Jun;97(11):4873-85. doi: 10.1007/s00253-012-4355-y. Epub 2012 Sep 1.
Cellobiose dehydrogenases (CDHs) are extracellular glycosylated haemoflavoenzymes produced by many different wood-degrading and phytopathogenic fungi. Putative cellobiose dehydrogenase genes are recurrently discovered by genome sequencing projects in various phylogenetically distinct fungi. The genomes from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina were screened for candidate cdh genes, and one and three putative gene models were evidenced, respectively. Two putative cdh genes were selected and successfully expressed for the first time in Aspergillus niger. CDH activity was measured for both constructions (CDHcc and CDHpa), and both recombinant CDHs were purified to homogeneity and subsequently characterised. Kinetic constants were determined for several carbohydrates including β-1,4-linked di- and oligosaccharides. Optimal temperature and pH were 60 °C and 5 for CDHcc and 65-70 °C and 6 for CDHpa. Both CDHs showed a broad range of pH stability between 4 and 8. The effect of both CDHs on saccharification of micronized wheat straw by an industrial Trichoderma reesei secretome was determined. The addition of each CDH systematically decreased the release of total reducing sugars, but to different extents and according to the CDH concentration. Analytical methods were carried out to quantify the release of glucose, xylose and gluconic acid. An increase of glucose and xylose was measured at a low CDHcc concentration. At moderated and high CDHcc and CDHpa concentrations, glucose was severely reduced with a concomitant increase of gluconic acid. In conclusion, these results give new insights into the physical and chemical parameters and diversity of basidiomycetous and ascomycetous CDHs. These findings also demonstrated that CDH drastically influenced the saccharification on a natural substrate, and thus, CDH origin, concentration and potential enzymatic partners should be carefully considered in future artificial secretomes for biofuel applications.
纤维二糖脱氢酶（CDHs）是由许多不同的木质素降解和植物病原真菌产生的细胞外糖基化血红素酶。通过各种不同系统发育真菌的基因组测序项目，经常发现假定的纤维二糖脱氢酶基因。筛选担子菌 Coprinopsis cinerea 和子囊菌 Podospora anserina 的基因组，分别鉴定出一个和三个假定的 cdh 基因模型。选择了两个假定的 cdh 基因，并首次在黑曲霉中成功表达。测定了两种构建体（CDHcc 和 CDHpa）的 CDH 活性，两种重组 CDH 均被纯化为均相并随后进行了表征。确定了几种碳水化合物的动力学常数，包括β-1,4 连接的二糖和寡糖。CDHcc 的最佳温度和 pH 值分别为 60°C 和 5，CDHpa 的最佳温度和 pH 值分别为 65-70°C 和 6。两种 CDH 在 4-8 之间均具有较宽的 pH 稳定性范围。测定了两种 CDH 对工业 Trichoderma reesei 分泌酶糖化微米化小麦秸秆的影响。添加每种 CDH 均系统地降低了总还原糖的释放，但释放程度不同，并且取决于 CDH 的浓度。进行了分析方法以定量测定葡萄糖、木糖和葡萄糖酸的释放。在低浓度的 CDHcc 下，测量到葡萄糖和木糖的增加。在适度和高浓度的 CDHcc 和 CDHpa 下，葡萄糖严重减少，同时葡萄糖酸增加。总之，这些结果为担子菌和子囊菌 CDH 的物理和化学参数和多样性提供了新的见解。这些发现还表明 CDH 极大地影响了天然底物的糖化，因此，在未来用于生物燃料应用的人工分泌酶中，应仔细考虑 CDH 的来源、浓度和潜在的酶伴侣。
