Cellobiose Dehydrogenase and a Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation by Neurospora crassa
Cellobiose Dehydrogenase and a Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation by Neurospora crassa
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DOI：
10.1021/cb200351y
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发表时间：
2011-12-01
影响因子：
4
通讯作者：
Marletta, Michael A.
中科院分区：
文献类型：
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作者：
Phillips, Christopher M.;Beeson, William T.;Marletta, Michael A.
木质纤维素生物质糖化酶的高成本是第二代生物燃料生产的主要障碍。利用遗传和生化技术的结合，我们报道了丝状真菌使用氧化酶来切割纤维素中的糖苷键。缺失粗草神经孢子菌的主要纤维素二氢酶编码基因cdh-1后，纤维素酶活性大幅降低，将嗜热分枝杆菌纯化的纤维素二氢酶添加到Delta cdh-1菌株中，纤维素酶活性提高了1.6- 2.0倍。在纯化的纤维素酶混合物中加入纤维素二糖脱氢酶没有刺激作用。我们发现，纤维素二糖脱氢酶通过将纤维素二糖的氧化与铜依赖的多糖单加氧酶（PMOs）的还原激活偶联，从而促进纤维素的降解，PMOs催化氧插入到毗邻糖苷键的C-H键中。这些PMOs中的三种被表征并显示出不同的区域特异性，导致氧化产物在葡聚糖链的还原端或非还原端被修饰。以前的模型认为氧化酶会产生随机攻击底物的活性氧，与之相反，这里的数据支持直接的、酶催化的纤维素氧化。纤维二糖脱氢酶和多糖单加氧酶相关的蛋白质在子囊菌和担子菌真菌中都被发现，这表明这种氧化纤维素降解模式可能在真菌界广泛存在。当添加到纤维素酶的混合物中时，这些蛋白质可以增强纤维素的糖化作用，这表明它们可以用于降低生物燃料生产的成本。
The high cost of enzymes for saccharification of lignocellulosic biomass is a major barrier to the production of second generation biofuels. Using a combination of genetic and biochemical techniques, we report that filamentous fungi use oxidative enzymes to cleave glycosidic bonds in cellulose. Deletion of cdh-1, the gene encoding the major cellobiose dehydrogenase of Neurospora crassa, reduced cellulase activity substantially, and addition of purified cellobiose dehydrogenases from M. thermophila to the Delta cdh-1 strain resulted in a 1.6- to 2.0-fold stimulation in cellulase activity. Addition of cellobiose dehydrogenase to a mixture of purified cellulases showed no stimulatory effect. We show that cellobiose dehydrogenase enhances cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of copper-dependent polysaccharide monooxygenases (PMOs) that catalyze the insertion of oxygen into C-H bonds adjacent to the glycosidic linkage. Three of these PMOs were characterized and shown to have different regiospecifities resulting in oxidized products modified at either the reducing or nonreducing end of a glucan chain. In contrast to previous models where oxidative enzymes were thought to produce reactive oxygen species that randomly attacked the substrate, the data here support a direct,enzyme-catalyzed oxidation of cellulose. Cellobiose dehydrogenases and proteins related to the polysaccharide monooxygenases described here are found throughout both ascomycete and basidiomycete fungi, suggesting that this model for oxidative cellulose degradation may be widespread throughout the fungal kingdom. When added to mixtures of cellulases, these proteins enhance cellulose saccharification, suggesting that they could be used to reduce the cost of biofuel production.