Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii.

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far.

The influence of different linker modifications on the catalytic activity and cellulose affinity of cellobiohydrolase Cel7A from Hypocrea jecorina.

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse.

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose.

Cel7A cellobiohydrolases perform processive hydrolysis on one strand of cellulose, which is threaded through the enzyme's substrate binding tunnel. The tunnel structure results from a groove in the catalytic domain, which is covered by a number of loops. These loops have been identified as potential targets for engineering of this industrially important enzyme family, but only few systematic studies on this have been made.

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases.

We measured hydrolytic rates of four purified cellulases in small increments of temperature (10-50 °C) and substrate loads (0-100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load.

A chromogenic assay for limit dextrinase and pullulanase activity.

A new chromogenic substrate to assay the starch debranching enzymes limit dextrinase and pullulanase is described. The 2-chloro-4-nitrophenyl glycoside of a commercially available branched heptasaccharide (Glc-maltotriosyl-maltotriose) was found to be a suitable specific substrate for starch debranching enzymes and allows convenient assays of enzymatic activities in a format suited for high-throughput analysis. The kinetic parameters of these enzymes toward the synthesized substrate are determined, and the selectivity of the substrate in a complex cereal-based extract is established.