Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases

In this study, we investigated the enzymatic hydrolysis of pretreated sugarcane bagasse using eight different enzymatic blends obtained from concentrated crude enzyme extracts produced by Penicillium funiculosum and Trichoderma harzianum as well as from the extracts in combination with a commercial enzymatic cocktail. The influence of different levels of biomass delignification, degree of crystallinity of lignicellulose, composition of enzymatic activities and BSA on enzymatic hydrolysis yields (HYs) was evaluated. Our X-ray diffraction studies showed that crystallinity of lignocellulose is not a key determinant of its recalcitrance toward enzymatic hydrolysis. In fact, under the experimental conditions of our study, an increase in crystallinity of lignocellulosic samples resulted in increased glucose release by enzymatic hydrolysis. Furthermore, under the same conditions, the addition of BSA had no significant effect on enzymatic hydrolysis. The most efficient enzyme blends were obtained by mixing a commercial enzymatic cocktail with P. funiculosum or T. harzianum cellulase preparations (HYs above 97%) followed by the concentrated extract of P. funiculosum alone (HY= 88.5%). Increased hydrolytic efficiencies appeared to correlate with having an adequate level of both beta-glucosidase and xylanase activities in the blends. (C) 2011 Elsevier Ltd. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

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.

Accelerated testing of mold growth on traditional and recycled book paper

The growth of molds on paper containing cellulose is a frequent occurrence when the level of relative air humidity is high or when books become wet due to water leaks in libraries. The aim of this study is to differentiate the bioreceptivity of different types of book paper for different fungi. Laboratory tests were performed with strains of Aspergillus niger, Cladosporium sp., Chaetomium globosum and Trichoderma harzianum isolated from books. Four paper types were evaluated: couche Men (offset), recycled and a reference paper containing only cellulose. The tests were carried out in chambers with relative air humidity of 95% and 100%. Mold growth was greatest in the tests at 100% relative humidity. Results of stereoscopic microscopy observation showed that Cladosporium sp. grew in 74% of these samples, A. niger in 75%, T. harzianum in 72% and C. globosum in 60%. In the chambers with 95% air humidity Cladosporium sp. grew in only 9% of the samples, A. niger in 1%, T harzianum in 3% and C globosum did not grow in any sample. The most bioreceptive paper was couche and the least receptive was recycled paper. The composition of the recycled paper, however, varies depending on the types of waste materials used to make it. (C) 2011 Elsevier Ltd. All rights reserved.

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.

Isolation of Brazilian marine fungi capable of growing on DDD pesticide

The fungi Aspergillus sydowii Ce15, Aspergillus sydowii Ce19, Aspergillus sydowii Gc12, Bionectria sp. Ce5, Penicillium miczynskii Gc5, Penicillium raistrickii Ce16 and Trichoderma sp. Gc1, isolated from marine sponges Geodia corticostylifera and Chelonaplysylla erecta, were evaluated for their ability to grow in the presence of DDD pesticide. Increasing concentrations of DDD pesticide, i.e., 5.0 mg (1.56 x 10(-12) mmol), 10.0 mg (3.12 x 10(-2) mmol) and 15.0 mg (4.68 x 10(-2) mmol) in solid and liquid culture media were tested. The fungi Trichoderma sp. Gc1 and Penicillium miczynskii Gc5 were able to grow in the presence of up to 15.0 mg of DDD, suggesting their potential for biodegradation. A 100% degradation of DDD was attained in liquid culture medium when Trichoderma sp. Gc1 was previously cultivated for 5 days and supplemented with 5.0 mg of DDD in the presence of hydrogen peroxide. However, the quantitative analysis showed that DDD was accumulated on mycelium and biodegradation level reached a maximum value of 58% after 14 days.