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.

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.

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.

Metal-buffered systems.

The design of a metal ion buffer system useful in a given enzymological application is subject to a number of different requirements. 1. The total concentration of added metal ion, Mt, should be large enough to damp out the effect of any adventitious quantities of the same metal ion and to overwhelm adventitious quantities of other metal ions. 2. The ratio of free to bound ligand should be high enough that the calculated ratio between the concentrations of free metal ion M and Mt will not be unduly sensitive to uncertainties in the values of metal-ligand stability constants. If possible, [Lt]/[Mt] should be large enough that the variation of free metal ion concentration, [M], with [Mt] will be effectively linear in the range of interest. 3. The concentrations of metal ion buffer species, both the free ligand and metal-ligand complexes, should be kept reasonably low in order to minimize the possibility of perturbation of the enzyme/metal ion equilibrium. The best design will be that which most successfully balances these sometimes opposing requirements.

A transition-state-analog inhibitor influences zinc-binding by Aeromonas aminopeptidase.

The transition-state-analog inhibitor, 1-butaneboronic acid, markedly enhances the uptake of one g-atom of Zn2+ ions from a metal ion buffer system by Zn-depleted Aeromonas aminopeptidase. In contrast, a substrate-analog inhibitor, n-valeramide, does not perturb the equilibrium between Zn2+ ions and the enzyme in a metal ion buffer system. These results establish a role for metal ions in the binding of 1-butaneboronic acid to Aeromonas amino-peptidase and strongly imply that a bound Zn2+ ion interacts directly with substrate during catalysis but not during initial binding of substrate.

Proteolytic specificity of hemorrhage toxin a isolated from western diamondback rattlesnake (Crotalus atrox) venom.

The proteolytic specificity of hemorrhagic toxin a from the venom of Crotalus atrox (western diamondback rattlesnake) has been investigated by using the oxidized B chain of bovine insulin and other peptides as substrates. The toxin appears highly specific for X--Leu bonds (cleaving the His10--Leu11, Ala14--Leu15, and Tyr16--Leu17 bonds), with no detectable activity against the Gly--Phe, Phe--Phe, Phe--Tyr, and Leu--Tyr bonds also present in the insulin B chain. The X--Leu bond of the peptides Tyr-Gly-Gly-Phe-Leu, Phe-Ala-Leu, and Ala-Leu was also cleaved. The toxin seems to be a strict endopeptidase, in that the cleavage of the two most susceptible bonds, Ala14--Leu15 and Tyr16--Leu17, are mutually exclusive; i.e., cleavage of either bond results in the other being too close to either the amino- or carboxyl-terminal of its respective fragment for the enzyme to be effective against it. The X--Met bond of Tyr-Gly-Gly-Phe-Met was cleaved, although a dipeptide Gly-Met was not hydrolyzed after 16 h of incubation. The substrates not hydrolyzed are furylacryloylglycyl-L-leucinamide, carbobenzoxy-L-glutamylglycine, carbobenzoxyglycyl-L-glutamic acid, benzoyl-L-arginine-p-nitroanilide, L-lysine-p-nitroanilide, (L-Ala)3-p-nitroanilide, Gly-Met, Gly-Phe-Phe, Gly-Gly-Ala, TAME, and ATEE. The absence of hydrolytic activity against the last two substrates indicates that hemorrhagic toxin a does not possess trypsin- or chymotrypsin-like activity.