Processive Endoglucanases Mediate Degradation of Cellulose by Saccharophagus degradans

Bacteria and fungi are thought to degrade cellulose through the activity of either a complexed or a noncomplexed cellulolytic system composed of endoglucanases and cellobiohydrolases. The marine bacterium Saccharophagus degradans 2-40 produces a multicomponent cellulolytic system that is unusual in its abundance of GH5-containing endoglucanases. Secreted enzymes of this bacterium release high levels of cellobiose from cellulosic materials. Through cloning and purification, the predicted biochemical activities of the one annotated cellobiohydrolase Cel6A and the GH5-containing endoglucanases were evaluated. Cel6A was shown to be a classic endoglucanase, but Cel5H showed significantly higher activity on several types of cellulose, was the highest expressed, and processively released cellobiose from cellulosic substrates. Cel5G, Cel5H, and Cel5J were found to be members of a separate phylogenetic clade and were all shown to be processive. The processive endoglucanases are functionally equivalent to the endoglucanases and cellobiohydrolases required for other cellulolytic systems, thus providing a cellobiohydrolase-independent mechanism for this bacterium to convert cellulose to glucose.The microbial degradation of cellulose is of interest due to applications in the sugar-dependent production of alternative biofuels (25). There are well-characterized cellulolytic systems of bacteria and fungi that employ multiple endo-acting glucanases and exo-acting cellobiohydrolases in the degradation of cellulose (12). For example, the noncomplexed cellulase system of the wood soft rot fungus Hypocrea jecorina (anamorph Trichoderma reesei), the source for most commercially available cellulase preparations, produces up to eight secreted β-1,4-endoglucanases (Cel5A, Cel5B, Cel7B, Cel12A, Cel45A, Cel61A, Cel61B, and Cel61C), two cellobiohydrolases (Cel6A and Cel7A), and several β-glucosidases (e.g., Bgl3A) (21). Cellobiohydrolases are critical to the function of these systems, as, for example, Cel7A comprises in excess of 50% of the cellulases secreted by this organism (11). Another well-characterized noncomplexed cellulase system is found in Thermobifida fusca, a filamentous soil bacterium that is a major degrader of organic material found in compost piles (32). This bacterium also secretes several endoglucanases and end-specific cellobiohydrolases to degrade cellulose (32). An alternative mechanism for degradation of cellulose is found in microorganisms producing complexed cellulolytic systems, such as those found in cellulolytic clostridia. In these microorganisms, several β-1,4-endoglucanases and cellobiohydrolases assemble on surface-associated scaffoldin polypeptides to form cellulose-degrading multiprotein complexes known as cellulosomes (2, 6). The unifying theme in both complexed and noncomplexed systems is the importance of cellobiohydrolases in converting cellulose and cellodextrins to soluble cellobiose.Recently, a complete cellulolytic system was reported to occur in the marine bacterium Saccharophagus degradans 2-40 (28, 31). This bacterium is capable of growth on both crystalline and noncrystalline celluloses as sole carbon sources and produces multiple glucanases that can be detected in zymograms of cell lysates (28). The genome sequence of this bacterium predicts that the cellulolytic system of this bacterium consists of 10 GH5-containing β-1,4-endoglucanases (Cel5A, Cel5B, Cel5C, Cel5D, Cel5E, Cel5F, Cel5G, Cel5H, Cel5I, and Cel5J), two GH9 β-1,4-endoglucanases (Cel9A and Cel9B), one cellobiohydrolase (Cel6A), five β-glucosidases (Bgl1A, Bgl1B, Bgl3C, Ced3A, and Ced3B), and a cellobiose phosphorylase (Cep94A) (28, 31). The apparent absence of a homolog to a scaffoldin in the genome sequence and to dockerin-like domains in the proposed glucanases suggests that this bacterium produces a noncomplexed cellulolytic system. Two unusual features of this cellulolytic system are the large number of GH5 endoglucanases and the presence of only one annotated cellobiohydrolase, Cel6A (28, 31). The apparent deficiency of cellobiohydrolases in this system raised the question as to the mechanism by which this bacterium degrades cellulose.To understand the mechanism for degradation of cellulose, the biochemical activities for the predicted cellobiohydrolase Cel6A and each of the GH5 glucanases predicted for the S. degradans cellulolytic system were evaluated. Cel6A exhibited properties of a classic endoglucanase, but three of the originally annotated endoglucanases, Cel5G, Cel5H, and Cel5J, were shown to be processive, forming cellobiose as the end product. Processive endoglucanases substitute for cellobiohydrolases in this system to play a major role in the degradation of cellulose.