A bacterial GH6 cellobiohydrolase with a novel modular structure.

Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn31,2) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a K m of 68.73 mg/ml and a V max of 1.73 U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.

New GH16 ß-glucanase from Paenibacillus barcinonensis BP-23 releases a complex pattern of mixed-linkage oligomers from barley glucan.

The gene coding for a lichenase from Paenibacillus barcinonensis BP-23, a powerful carbohydrate-degrading strain, was obtained using a genome walking strategy and expressed in Escherichia coli for further characterization. The amino acid sequence deduced from lic16A revealed that the lichenase is a single-domain enzyme belonging to the GH16 family. Purified recombinant Lic16A showed exclusive activity on ß-1,3-1,4-glucans, showing a Km of 16.88 mg/mL and a Vmax of 266.09 U/mg using lichenan as a substrate. Lic16A was stable at 55 °C for at least 3 H in moderate pH conditions. Thin-layer chromatography analysis showed that the enzyme released a complex mixture of hydrolysis products, which consisted of different length oligosaccharides of intermediate mobility among cellooligomers. The health benefits of ß-glucans's consumption and the increased interest for the use of their oligomers as prebiotics add interest to the study of Lic16A for the production of ß-glucan-derived oligosaccharides and the evaluation of their biotechnological potential. This is the first report on ß-1,3-1,4-glucanase produced by P. barcinonensis.

Structural analysis of glucuronoxylan-specific Xyn30D and its attached CBM35 domain gives insights into the role of modularity in specificity.

Glucuronoxylanase Xyn30D is a modular enzyme containing a family 30 glycoside hydrolase catalytic domain and an attached carbohydrate binding module of the CBM35 family. We present here the three-dimensional structure of the full-length Xyn30D at 2.4 Å resolution. The catalytic domain folds into an (α/ß)8 barrel with an associated ß-structure, whereas the attached CBM35 displays a jellyroll ß-sandwich including two calcium ions. Although both domains fold in an independent manner, the linker region makes polar interactions with the catalytic domain, allowing a moderate flexibility. The ancillary Xyn30D-CBM35 domain has been expressed and crystallized, and its binding abilities have been investigated by soaking experiments. Only glucuronic acid-containing ligands produced complexes, and their structures have been solved. A calcium-dependent glucuronic acid binding site shows distinctive structural features as compared with other uronic acid-specific CBM35s, because the presence of two aromatic residues delineates a wider pocket. The nonconserved Glu(129) makes a bidentate link to calcium and defines region E, previously identified as specificity hot spot. The molecular surface of Xyn30D-CBM35 shows a unique stretch of negative charge distribution extending from its binding pocket that might indicate some oriented interaction with its target substrate. The binding ability of Xyn30D-CBM35 to different xylans was analyzed by affinity gel electrophoresis. Some binding was observed with rye glucuronoarabinoxylan in presence of calcium chelating EDTA, which would indicate that Xyn30D-CBM35 might establish interaction to other components of xylan, such as arabinose decorations of glucuronoarabinoxylan. A role in depolymerization of highly substituted chemically complex xylans is proposed.

Crystallization and preliminary X-ray diffraction analysis of Xyn30D from Paenibacillus barcinonensis.

Xyn30D, a new member of a recently identified group of xylanases, has been purified and crystallized. Xyn30D is a bimodular enzyme composed of an N-terminal catalytic domain belonging to glycoside hydrolase family 30 (GH30) and a C-terminal family 35 carbohydrate-binding domain (CBM35) able to bind xylans and glucuronic acid. Xyn30D shares the characteristic endo mode of action described for GH30 xylanases, with the hydrolysis of the ß-(1,4) bonds of xylan being directed by α-1,2-linked glucuronate moieties, which have to be placed at the -2 subsite of the xylanase active site. Crystals of the complete enzyme were obtained and a full data set to 2.3 Šresolution was collected using a synchrotron X-ray source. This represents the first bimodular enzyme with the domain architecture GH30-CBM35. This study will contribute to the understanding of the role that the different xylanases play in the depolymerization of glucuronoxylan.

Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module.

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (ß/α)(8) barrel linked to a side-associated ß-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side ß-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

Characterization of a family GH5 xylanase with activity on neutral oligosaccharides and evaluation as a pulp bleaching aid.

A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.