Type-dependent action modes of TtAA9E and TaAA9A acting on cellulose and differently pretreated lignocellulosic substrates.

BACKGROUND: Lytic polysaccharide monooxygenase (LPMO) is a group of recently identified proteins that catalyze oxidative cleavage of the glycosidic linkages of cellulose and other polysaccharides. By utilizing the oxidative mode of action, LPMOs are able to enhance the efficiency of cellulase in the hydrolysis of cellulose. Particularly, auxiliary activity family 9 (AA9) is a group of fungal LPMOs that show a type-dependent regioselectivity on cellulose in which Types 1, 2, and 3 hydroxylate at C1, C4, and C1 and C4 positions, respectively. In this study, we investigated comparative characteristics of TtAA9E from Thielavia terrestris belonging to Type 1 and TaAA9A from Thermoascus aurantiacus belonging to Type 3 on cellulose and pretreated lignocellulose. RESULTS: From product analysis, TtAA9E dominantly generated oligosaccharides with an aldonic acid form, which is an evidence of C1 oxidation, while TaAA9A generated oligosaccharides with both aldonic acid and 4-ketoaldose forms, which is evidence of C1 and C4 oxidations, respectively. For hydrolysis of cellulose (Avicel) by cellulase, higher synergism was observed for TtAA9E than for TaAA9A. For hydrolysis of pretreated lignocellulose using rice straw, synergistic behaviors of TtAA9E and TaAA9A were different depending on the pretreatment of rice straw. Specifically, on acid-pretreated rice straw, TtAA9E showed a higher synergism than TaAA9A while on alkali-pretreated rice straw, TaAA9A showed a higher synergism than TtAA9E. CONCLUSIONS: We show type-dependent action modes of TtAA9E and TaAA9A for cellulose oxidation together with substrate-dependent synergistic hydrolysis of cellulosic substrates. The results obtained from this study indicate the different behaviors of AA9s on cellulose and pretreated lignocellulose, suggesting a selection of AA9 proteins specific to substrates is required for industrial utilization.

Three-dimensional structures of two heavily N-glycosylated Aspergillus sp. family GH3 ß-D-glucosidases.

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant ß-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. ß-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 ß-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Šresolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley ß-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy (4)C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal ß-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by ß-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

The three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5†Šresolution.

The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of the Aspergillus fumigatus CBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing three-dimensional insight into this family of industrially relevant biocatalysts.

New enzyme insights drive advances in commercial ethanol production.

Innovations at a small scale through enzyme discovery in the laboratory can have large scale impacts when rolled out in an industrial process, and this is evidenced in recent advances for commercial ethanol production. In the starch to ethanol processes, new enzyme product launches squeeze even more value from an already efficient process, as evidenced in new use of proteases for oil release and cellulases for downstream processing and ethanol yield. As for biomass to ethanol, diverse new thermophilic enzymes, expansins and auxiliary activity (AA) collections are growing rapidly. Our mechanistic understanding of the functions of AA family 9, cellulose binding modules, and cellulase/xylanase synergy will lead to continued improvements in overall enzymatic conversion, thus reducing cost for cellulosic ethanol (or other biofuel) production.

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum.

A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60 degrees C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-beta-D-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-13C6 as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.

A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry.

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. beta-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few beta-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover beta-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and beta-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as beta-glucosidases was confirmed by tandem mass spectrometry. Two novel beta-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel beta-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed beta-glucosidase showed far superior heat stability to the previously characterized beta-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of beta-glucosidases in a high throughput fashion.