Irreversible transformations of native celluloses, upon exposure to elevated temperatures.

Current research, basic and applied, assumes that observed recalcitrance of celluloses is an inherent characteristic associated with their state of aggregation in their native state; it is thought that processes of isolation remove other components of plant cell walls leaving the celluloses unchanged, even though elevated temperatures are routinely used during isolation. Since temperature elevation is known to influence the structures of all polymers, it is important to explore its influence on the character of isolated celluloses, almost always assumed to be still in their native state. Deuterium exchange is a measure of accessibility of reactive sites in celluloses. We report significant reduction in accessibility to deuterium and other probe molecules for celluloses isolated at ambient temperature and then exposed to elevated temperatures. Our results indicate that native celluloses, which are highly ordered biological structures, are irreversibly transformed and develop polymeric semi-crystalline character upon isolation at elevated temperatures.

Cost effectiveness of neoadjuvant chemotherapy in locally advanced operable head and neck cancer followed by surgery and postoperative radiotherapy: a Markov model-based decision analysis.

INTRODUCTION: Head and neck carcinomas are among the most frequent tumor diseases and, because of different multimodal therapy options, cause enormous costs. For this reason, we examined whether in operable advanced head and neck carcinomas, neoadjuvant induction chemotherapy is cost effective in comparison with surgery followed by postoperative radio(chemo)therapy. MATERIAL AND METHODS: A Markov model was developed with paclitaxel, cisplatin and fluorouracil as induction therapy. The legal health insurance in Germany was chosen for cost perspectives, and a willingness-to-pay limit at EUR 38,000 was set. RESULTS: Total costs for surgery with postoperative radiochemotherapy amounted to EUR 13,999. Prior induction chemotherapy raised the costs to EUR 17,377, with a higher effectiveness by 0.1 years of life. Costs per year of life gained are EUR 33,780. The incremental cost effectiveness ratio (ICER) with variations in side effects for surgery and postoperative chemotherapy amounted to between EUR 31,520 and 36,050. With variations in side effects for induction chemotherapy, the ICER amounted to EUR 30,060-37,520. The Monte Carlo simulation disclosed cost effectiveness for 55.4% of the patients; for 44.6%, there was no cost effectiveness. CONCLUSION: The Markov-modeled cost effectiveness analysis indicates that with operable head and neck tumors, induction therapy with paclitaxel, cisplatin and fluorouracil is cost effective.

Subtype-specific statistical causalities in parkinsonian tremor.

Tremor is one of the cardinal symptoms of Parkinson's disease. Up to now, however, its pathophysiology remains poorly understood. Previously, oscillatory coupling at tremor frequency between the subthalamic nucleus und affected muscles was shown. In these studies, however, causality of coupling could not be demonstrated. Thus, we analyzed the statistical causality between intraoperatively recorded local field potentials in the subthalamic area and affected arm muscles during tremor episodes, using squared partial directed coherence, a recently developed causality measure. The analysis identified differential statistical causality patterns for Parkinson's disease patients of the akinetic-rigid subtype during tremor episodes (n=6) versus patients of the tremor-dominant subtype (n=8): for the akinetic-rigid Parkinson's disease patients significantly more cases of the subthalamic region were found to be statistically causal for electromyographic-tremor activity, a result in accordance with the standard basal ganglia model. In contrast, for the tremor-dominant patients, significantly more instances of electromyographic tremor activity turned out to be causal for activity of the subthalamic region. Furthermore, the clinical effective stimulation site coincided with the location of most input causalities from the periphery in seven out of eight tremor-dominant patients. The data suggest that, although tremor activity in tremor-dominant and akinetic-rigid Parkinson's disease patients was clinically similar, statistical causality between tremor electromyogram (EMG) and the subthalamic nucleus was fundamentally different. Therefore, we hypothesize different pathophysiological mechanisms to underlie the generation of tremor in the two subtypes of Parkinson's disease.

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2).

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.

Energetics for displacing a single chain from the surface of microcrystalline cellulose into the active site of Acidothermus cellulolyticus Cel5A.

A series of molecular mechanics calculations were used to analyze the energetics for moving a single polysaccharide chain from the surface of microcrystalline cellulose into the binding cleft of the Cel5A cellulase from Acidothermus cellulolyticus. A build-up procedure was used to model the placement of a 12-residue oligosaccharide chain along the surface of the enzyme, using as a guide the four residues of the tetrasaccharide substrate co-crystallized with the protein in the crystallographic structure determination. The position of this 12-residue oligosaccharide was used to orient the enzyme properly above two different surfaces of cellulose 1beta, the (1,0,0) and the (1,1,0) faces of the crystal. Constrained molecular dynamics simulations were then used to pull a target chain directly below the enzyme up out of the crystal surface and into the binding groove. The energetics for this process were favorable for both faces, with the step face being more favorable than the planar face, implying that this surface could be hydrolyzed more readily.

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to "fingerprint" analysis by high-resolution 2-D gel electrophoresis. Forty-five spots from 11 x 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

Indications for a novel muscular dystrophy pathway. gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin.

gamma-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although gamma-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of gamma-filamin. The two Ig-like domains of the recently described alpha-actinin-binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of gamma-filamin may be indirectly anchored to alpha-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.

Enzymes, Energy, and the Environment: A Strategic Perspective on the U.S. Department of Energy's Research and Development Activities for Bioethanol.

For well over one hundred years, researchers around the world have pursued ways to make ethanol from biomass such as wood, grasses, and waste materials. To distinguish it from ethanol made from starch and sugars in traditional agricultural crops, we refer to ethanol made from biomass as "bioethanol." The effort to develop bioethanol technology gained significant momentum in the late 1970s as a result of the energy crises that occurred in that decade. This article briefly reviews the broader history of bioethanol technology development. With this as a background, we focus our attention on the strategic thinking behind the U.S. Department of Energy's Bioethanol Program, which envisions remarkable advances in cellulase enzyme research and as the basis for significant future process cost reductions.

Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris.

Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS-PAGE gels. Carbohydrate content determination and SDS-PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild-type enzyme. More seriously, the yeast-expressed enzyme showed non-wild-type secondary structure by circular dichroism. We conclude that P. pastoris may not serve as an adequate host for the site-directed mutagenesis of T. reesei CBH I.

Hydrolysis of cellulose using ternary mixtures of purified cellulases.

The saccharification of microcrystalline cellulose by reconstituted ternary mixtures of purified cellulases (one endoglucanase and two cellobiohydrolases) has been studied over the entire range of mixture compositions. Ternary plots are used to compare the performance of five synthetic mixtures drawn from the cellulase systems of Acidothermus cellulolyticus, Trichoderma reesei, Thermomonospora fusca, and Thermotoga neapolitana. Results reveal that at least one synthetic mixture utilizing enzymes from three different organisms delivers performance competitive with that of a "native" (i.e., co-evolved) ternary system drawn exclusively from T. reesei. This heterologous system, consisting of the endoglucanase E1 from A. cellulolyticus and the exoglucanases CBHI from T. reesei and E3 from T. fusca, is forgiving from the system-design point of view, in that it delivers high saccharification rates over a wide range of mixture compositions.

Hydrolysis of cellulose using ternary mixtures of purified celluloses.

The saccharification of microcrystalline cellulose by reconstituted ternary mixtures of purified cellulases (one endoglucanase and two cellobiohydrolases) has been studied over the entire range of mixture compositions. Ternary plots are used to compare the performance of five synthetic mixtures drawn from the cellulase systems of Acidothermus cellulolyticus, Trichoderma reesei, Thermomonospora fusca, and Thermotoga neapolitana. Results reveal that at least one synthetic mixture utilizing enzymes from three different organisms delivers performance competitive with that of a "native" (i.e., co-evolved) ternary system drawn exclusively from T. reesei. This heterologous system, consisting of the endoglucanase El from A. cellulolyticus and the exoglucanases CBHI from T. ressei and E3 from T. fusca, is forgiving from the system-design point of view, in that it delivers high saccharification rates over a wide range of mixture compositions.

Polysaccharide hydrolase folds diversity of structure and convergence of function.

Polysaccharide glycosyl hydrolases are a group of enzymes that hydrolyze the glycosidic bond between carbohydrates or between a carbohydrate and a noncarbohydrate moiety. Here we illustrate that traditional schemes for grouping enzymes, such as by substrate specificity or by organism of origin, are not appropriate when thinking of structure-function relationships and protein engineering. Instead, sequence comparisons and structural studies reveal that enzymes with diverse specificities and from diverse organisms can be placed into groups among which mechanisms are largely conserved and insights are likely to be transferrable. In particular, we illustrate how enzymes have been grouped using protein sequence alignment algorithms and hydrophobic cluster analysis. Unfortunately for those who seek to improve cellulase function by design, cellulases are distributed throughout glycosyl hydrolase Families 1,5,6,7,9, and 45. These cellulase families include members from widely different fold types, i.e., the TIM-barrel, betaalphabeta-barrel variant (a TIM-barrel-like structure that is imperfectly superimposable on the TIM-barrel template), beta-sandwich, and alpha-helix circular array. This diversity in cellulase fold structure must be taken into account when considering the transfer and application of design strategies between various cellulases.

Use of a new membrane-reactor saccharification assay to evaluate the performance of celluloses under simulated SSF conditions : effect on enzyme quality of growing Trichoderma reesci in the presence of targeted lignocellulosic substrate.

A new saccharification assay has been devised, in which a continuously buffer-swept membrane reactor is used to remove the solubilized saccharification products, thus allowing high extents of substrate conversion without significant inhibitory effects from the buildup of either cellobiose or glucose. This diafiltration saccharification assay (DSA) can, therefore, be used to obtain direct measurements of the performance of combinations of cellulase and substrate under simulated SSF conditions, without the saccharification results being complicated by factors that may influence the subsequent fermentation step. This assay has been used to compare the effectiveness of commercial and special in-house-produced Trichoderma reesei cellulase preparations in the saccharification of a standardized microcrystalline (Sigmacell) substrate and a dilute-acid pretreated lignocellulosic substrate. Initial results strongly suggest that enzyme preparations produced in the presence of the targeted lignocellulosic substrate will saccharify that substrate more effectively. These results call into question the widespread use of the "filter paper assay" as a reliable predictor of enzyme performance in the extensive hydrolysis of substrates that are quite different from filter paper in both physical properties and chemical composition.

Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose.

The crystal structure of the catalytic domain of the thermostable endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose has been solved by multiple isomorphous replacement and refined at 2.4 A resolution to an R-factor of 0.18 (Rfree = 0.24). E1cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)8 barrel, which constitutes a prototype for family 5-subfamily 1 cellulases. The cellotetraose molecule binds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conserved in family 5 enzymes are involved in recognition of the glycosyl group attacked during cleavage. Whereas only three residues are conserved in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from which the name is derived), structural comparisons show that all eight residues conserved in family 5 have functional equivalents in the other 4/7 superfamily members, strengthening the case that mechanistic details are conserved throughout the superfamily. On the basis of the structure, a detailed sequence of physical steps of the cleavage mechanism is proposed. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pKa of the acid/base for the glycosylation and deglycosylation half-reactions. Finally, purely structural based comparisons are used to show that significant differences exist in structural similarity scores resulting from different methods and suggest that caution should be exercised in interpreting such results in terms of implied evolutional relationships.

Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli.

The process of converting lignocellulosic biomass to ethanol via fermentation depends on developing economic sources of cellulases. Trichoderma reesei cellobiohydrolase (CBH) I is a key enzyme in the fungal cellulase system; however, specific process application requirements make modification of the enzyme by site-directed mutagenesis (SDM) an attractive goal. To undertake SDM investigations, an efficient, cellulase-free host is required. To test the potential of Escherichia coli as a host, T. reesei CBH I cDNA was expressed in E. coli strain GI 724 as a C-terminal fusion to thermostable thioredoxin protein. Full-length expression of CBH I was subsequently verified by molecular weight, Western blot analysis, and activity on soluble substrates.

Purification and characterization of an acetyl esterase from Aspergillus niger.

Optimized acetyl esterase enzyme production conditions using Aspergillus niger ATCC 10864 in 14-L fermentation jars were determined to be 33 degrees C, 1.5 vvm aeration, and 300 rpm agitation without pH control. The acetyl esterase was purified by precipitation in 60-80% saturation in ammonium sulfate. The pellet was applied directly to a Pharmacia high-load Phenyl Sepharose column for hydrophobic interaction chromatography and purified to homogeneity in two steps. Stability and kinetic characteristics of the acetyl esterase were determined over a pH range of 4.0-7.5 and from 4 to 45 degrees C. At temperatures > 25 degrees C, stability was superior at pH values < 5.0. The temperature activity optimum was 35 degrees C, and the pH optimum was 7.0. The Vmax was determined to be 46,700 U/mg protein, and the Km was 0.023M p-nitrophenyl acetate at pH 6.5 in 0.2M phosphate buffer at 35 degrees C. The mol wt of the enzyme was 35,000 dalton by size-exclusion chromatography and SDS gel electrophoresis. The N-terminal amino acid sequence and the glycosylation composition were also determined.

Isolation and characterization of two forms of beta-D-glucosidase from Aspergillus niger.

beta-D-glucosidase purified from commercial preparations of clarified culture broth of Aspergillus niger (Novo SP188) was shown to elute as two distinct species during analytical anion-exchange chromatography (AEC). However, the two enzyme forms behaved identically on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), high-performance size-exclusion chromatography (HPSEC), and isoelectric focusing. Also, the N-terminal amino acid sequence, amino acid composition, fingerprint of tryptic-digest peptides, circular dichroism spectra, and reaction kinetics appear identical for these forms. This feature of the A. niger enzyme is distinctly different from beta-D-glucosidase isozymes reported from other sources, where multiple forms tend to differ in molecular weight and/or isoelectric pH. Michaelis-Menten kinetic analysis also gave comparable results for the two forms. The distinct behavior on AEC was explained by considering the differences in N-linked carbohydrates liberated from both species following treatment with endoglycosidase H or F.