Hydrolytic Activity of Free and Immobilized Cellulase

Cellulase is an enzymatic complex which synergically promotes the degradation of cellulose to glucose. The adsorption behavior of cellulase from Trichoderma reesei onto Si wafers or amino-terminated surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM) as a function of temperature. Upon increasing temperature from (24 +/- 1) to (60 +/- 1) degrees C, adsorption of cellulase became faster and more pronounced and the mean roughness of cellulase adsorbed layers increased. In the case of cellulase adsorbed onto Si wafers, Arrhenius`s plot allowed us to estimate the adsorption energy as 24.2 kJ mol(-1). The hydrolytic activity of free cellulase and cellulase immobilized onto Si wafers was tested using cellulose dispersions as substrates. The incubation temperature ranged from (37 +/- 1) to (60 +/- 1) degrees C. The highest efficiency was observed at (60 +/- 1) degrees C. The amount of glucose produced by free cellulase was similar to 20% higher than that obtained from immobilized cellulase. However, immobilizing cellulase onto Si wafers proved to be advantageous because they could be reused six times while retaining their original activity level. Such an effect was attributed to surface hydration, which prevents enzyme denaturation. The hydrolytic activity of cellulase immobilized onto amino-terminated surfaces was slightly lower than that observed for cellulase adsorbed onto Si wafers, and reuse was not possible.

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 angstrom resolution and 15.81% (R(free) = 19.2%) at 1.85 angstrom resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity. (C) 2008 Elsevier Ltd. All rights reserved.