Processive endoglucanases mediate degradation of cellulose by Saccharophagus degradans

Abstract

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.

FIG. 1.

Zymogram of S. degradans GH5 glucanase activities. After fractionation by SDS-PAGE and renaturing, retained barley glucan substrate was stained using Congo red after a 12-h digestion. Zones of clearing represent glucanase activity. Similar results were obtained for zymograms containing HE-cellulose. The expected molecular mass of each polypeptide as cloned is shown in parentheses under each lane label. Letters A through J refer to the Cel5 enzyme. With the exceptions of Cel5E and Cel5J, the expression of each protein in the Rosetta 2 (DE3) host appeared to be equivalent in Coomassie blue-stained gels, as the peak areas of each expressed protein were similar. To resolve the activity of polypeptides in the zymograms, the samples were diluted. The amount of protein in each well was equivalent to that in the original cell culture, as follows: Cel5A, 100 nl; Cel5B, 1,000 nl; Cel5C, 7,000 nl; Cel5D, 7,000 nl; Cel5E, 1,000 nl; Cel5F, 100 nl; Cel5G, 1 nl; Cel5H, 10 nl; Cel5I, 7,000 nl; and Cel5J, 10 nl. Precision Plus molecular mass markers (Bio-Rad, Hercules, CA) were used as molecular mass markers.

FIG. 2.

Effect of endo- and exoglucanases on CMC viscosity. S. degradans Cel5H (♦), T. fusca Cel9B (▪), and T. fusca Cel6B (▴) were added to a 1% CMC solution. The viscosity was measured as a function of time as described in Materials and Methods. The viscosity is given in centipoise (cP) units.

FIG. 3.

Products formed by S. degradans Cel5H activity. Controls: E, enzyme alone; A, Avicel alone; FP, filter paper alone; and SC, PASC alone. Products formed by the activity of purified Cel5H on the indicated substrate (A, Avicel; FP, filter paper; and SC, PASC) are shown. For both the control and the digestion trials, reaction mixtures were incubated at 50°C for 16 h. Two-microliter aliquots were spotted on Silica Gel G plates and resolved using a nitromethane-propanol-water solvent system as described in Materials and Methods. The markers G1 to G4 represent the migration of glucose, cellobiose, cellotriose, and cellotetraose, respectively. The time courses of products released by the activity of S. degradans Cel5H on PASC are shown. The reaction conditions were as described in Materials and Methods and the products resolved as described above. Time is indicated in minutes.

FIG. 4.

Processivity of Cel5H activity. Purified Cel5H was incubated with filter paper for the indicated times, and products formed as reducing sugar were determined as described in Materials and Methods. Diamonds (top line) indicate soluble reducing sugar detected (μmol cellobiose). Squares (bottom line) indicate insoluble reducing sugar (μmol glucose). Linear trend lines were calculated using y = mx. For release of soluble reducing sugar, m is 0.0108, with a goodness of fit R² value of 0.97. For formation of insoluble reducing sugar, m is 0.0020 and R² is 0.94.

FIG. 5.

Phylogenetic analysis of S. degradans GH5 domains and their processivity on filter paper. The sequences of the GH5 domains found in the S. degradans glucanases identified by Taylor et al. (28), together with the closest homologs identified in another organism by BLAST, were extracted and subjected to nearest-neighbor analysis as described in Materials and Methods. The resulting phylogenetic tree and the domain organization of each enzyme as predicted by the SMART algorithm are shown. The larger gray-filled boxes represent the location of the GH5 domain in each polypeptide. The red triangles denote the location of carbohydrate binding modules. The processivities of the indicated enzymes were determined as described for Table 4 and are shown in the embedded tables. The enzymes included in the upper-right embedded table have processivity values typical of classic endoglucanases, whereas the enzymes included in the lower-left embedded table have processivity values in excess of 4. The GenBank accession numbers for the genes, with the closest homologs shown in parentheses, are as follows: for the Cel5A-N gene, YP_435061 (endoglucanase [Hahella chejuensis KCTC 2396]); for the Cel5A-C gene, YP_528706 (Sde_3237 and Cel5H); for the Cel5B gene, ZP_00510594 (glycoside hydrolase family 5, Clostridium cellulosome enzyme dockerin type I, and carbohydrate binding domain family 11 [Clostridium thermocellum ATCC 27405]); for the Cel5C gene, ZP_01246425 (glycoside hydrolase family 5 [Flavobacterium johnsoniae UW101]); for the Cel5D gene, ZP_01115721 (endoglucanase family 5 [Reinekea sp. strain MED297]); for the Cel5E gene, ABD81750 (Sde_2490 and Cel5B); for the Cel5F gene, ABA02176 (Cellvibrio japonicus Ueda107); for the Cel5G gene, YP_528706 (Sde_3237 and Cel5H); for the Cel5H gene, YP_528708 (Sde_3239 and Cel5G); for the Cel5I gene, ZP_01113981 (endo-1,4-beta-glucanase [Reinekea sp. strain MED297]); and for the Cel5J gene, YP_435061 (endoglucanase [Hahella chejuensis KCTC 2396]).