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.

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.

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.

Anaerobic digestion of lignocellulosic biomass and wastes. Cellulases and related enzymes.

Anaerobic digestion represents one of several commercially viable processes to convert woody biomass, agricultural wastes, and municipal solid wastes to methane gas, a useful energy source. This process occurs in the absence of oxygen, and is substantially less energy intensive than aerobic biological processes designed for disposal purposes. The anaerobic conversion process is a result of the synergistic effects of various microorganisms, which serve as a consortium. The rate-limiting step of this conversion process has been identified as the hydrolysis of cellulose, the major polymeric component of most biomass and waste feedstocks. Improvements in process economics therefore rely on improving the kinetic and physicochemical characteristics of cellulose degrading enzymes. The most thoroughly studied cellulase enzymes are produced by aerobic fungi, namely Trichoderma reesei. However, the pH and temperature optima of fungal cellulases make them incompatible for use in anaerobic digestion systems, and the major populations of microorganisms involved in cellulase enzyme production under anaerobic digestion conditions are various bacterial producers. The current state of understanding of the major groups of bacterial cellulase producers is reviewed in this paper. Also addressed in this review are recently developed methods for the assessment of actual cellulase activity levels, reflective of the digester "hydrolytic potential," using a series of detergent extractive procedures.

Effect of Actinomyces pyogenes and gram-negative anaerobic bacteria on the development of bovine pyometra.

Fifteen lactating Holstein cows were used in a trial designed to evaluate the effectiveness of intrauterine inoculation (challenge) of Actinomyces pyogenes (A) alone or in combination with Fusobacterium necrophorum (F) and Bacteroides melaninogenicus (B) to induce pyometra. Cows were assigned to one of five groups: A (n = 3), AB (n = 3), AF (n = 3), ABF (n = 3) or C (control, broth medium alone; n = 3). All cows exhibited estrus 12 or 13 d prior to challenge (Day 0=first day of challenge). During the prechallenge period, the reproductive tract of each cow was palpated per rectum and uterine fluid aspirates for culture and uterine biopsies were also obtained. All cows received an intravenous injection of 5,000 IU of human chorionic gonadotropin (hCG; Day 5) and an intrauterine infusion of 40 ml of 0.7% iodine solution (Day 1). Cows were then inoculated on Days 0, 1 and 2 of the experiment. Sequential palpations of the reproductive tracts, samples of uterine fluid for culture and uterine biopsies were performed for a total of 30 d after the first inoculation. A cow was diagnosed as having pyometra when purulent uterine fluid and a corpus luteum were detected by palpation per rectum. The number of cows that developed pyometra in Group A was 2 of 3, in Group AB 3 of 3, in Group AF 3 of 3, in Group ABF 3 of 3 and in Group C 0 of 3. Cows with pyometra did not exhibit estrus. In 7 of 11 cows, pyometra persisted for more than 21 d. In cows that developed pyometra, the same species of bacteria infused into the uterus were usually recovered one or more times during the postchallenge period. When clinical pyometra was diagnosed, histologic endometritis was invariably present. Histologic endometritis and concurrent isolation of A . pyogenes alone or A . pyogenes with gram-negative anaerobic bacteria occurred in 91.7% of samples during the postchallenge period. Regardless of bacterial treatment, gram-negative anaerobic bacteria were frequently isolated with A . pyogenes during this period.

Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals.

Severe hemorrhagic necrotizing enterocolitis was determined to be the cause of death for 4 foals. Toxigenic Clostridium difficile was isolated form the intestine of each foal, and large, gram-positive, rod-shaped bacteria lined the surface of necrotic villi. This finding of toxigenic C difficile associated with enteritis in foals adds another possible cause to the list of infectious agents that should be considered when evaluating foals with enteritis. Definitive diagnosis requires a thorough diagnostic evaluation, including procedures that will identify the organism and demonstrate its toxigenicity.

Evaluation of Quantum II microbiology system for identification of gram-negative bacteria of veterinary origin.

The ability of a rapid, semiautomated bacterial identification system, the Quantum II microbiology system (Abbott Laboratories, Irving, Tex.), to accurately identify gram-negative bacteria from veterinary sources was evaluated. A total of 378 isolates were tested, including 298 organisms in the family Enterobacteriaceae and strains representing Acinetobacter sp., Aeromonas sp., Flavobacterium sp., Pasteurella multocida, Plesiomonas sp., and Pseudomonas spp. Of these isolates, 333 (88.1%) were correctly identified, 20 (5.3%) were not identified, 10 (2.6%) were incorrectly identified at the genus level, and 15 (4.0%) were incorrectly identified at the species level. The Quantum II system correctly identified 268 (89.9%) of the isolates of Enterobacteriaceae and 65 (81.3%) of the nonenteric isolates. P. multocida was not identified correctly, and some nonenteric gram-negative bacteria of clinical significance in veterinary medicine are not included in the data base. The Quantum II system provided an accurate identification system for isolates of Enterobacteriaceae but had limited usefulness for the identification of other gram-negative bacteria of clinical significance in veterinary medicine.

Isolation of Clostridium difficile and detection of cytotoxin in the feces of diarrheic foals in the absence of antimicrobial treatment.

Clostridium difficile was isolated from the feces of 27 of 43 diarrheic foals (63%), and cytotoxin was detected in feces from 28 diarrheic foals (65%). The foals had not received any antimicrobial treatment before the onset of diarrhea. C. difficile was not isolated from feces of 18 normal foals without diarrhea and 62 adult horses (P less than 0.005). This finding of C. difficile and its toxins in association with diarrhea in foals adds another possible cause to the list of infectious agents which may cause diarrhea in foals.

Anaerobic bacterial infections and response to treatment in dogs and cats: 36 cases (1983-1985).

Anaerobic bacteria have been increasingly implicated as important pathogens in animals. To determine the prevalence of anaerobic bacterial infection, the results of anaerobic bacteriologic culture of 599 specimens obtained from dogs and cats hospitalized at the Colorado State University Veterinary Teaching Hospital were reviewed. Obligate anaerobic bacteria were isolated from 35% of properly submitted specimens; Bacteroides spp and Fusobacterium spp were the organisms most commonly isolated. Infections most often containing anaerobes were abscesses, pleuropulmonary infections, and abdominal infections. A complication rate of 28% was observed with anaerobic bacterial infections; failure to initially treat with an effective antibiotic increased the rate of recurrence of infection.

Evaluation of the RapID-ANA system for identification of anaerobic bacteria of veterinary origin.

This study evaluated the ability of the RapID-ANA system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) to accurately identify a spectrum of freshly isolated veterinary anaerobes. A total of 183 isolates were tested and included 7 Actinomyces spp., 53 Bacteroides spp., 32 Clostridium spp., 2 Eubacterium spp., 65 Fusobacterium spp., 1 Peptococcus spp., 22 Peptostreptococcus spp., and 1 Propionibacterium spp. All isolates were initially identified by conventional biochemical testing and gas-liquid chromatography of short-chain fatty acid metabolites. Additional tests were performed as required by the RapID-ANA system. Of these isolates, 81.4% were correctly identified to the genus level, including 59.6% to the species level, 14.2% were incorrectly identified at the genus level, and 4.4% were not identified. Initially, 20.2% of the strains were not identified because the microcodes were not in the code book. The majority of the incorrect identifications were caused by the misidentification of Fusobacterium spp. as Bacteroides spp. Errors also occurred when veterinary anaerobes not included in the data base were assigned an identification from the existing data base. The RapID-ANA system appears to be a promising new method for rapid identification of veterinary anaerobes; however, further evaluation with an extended data base is needed before the system can accurately identify all clinically significant anaerobes.

Aspects of bacteriology and endocrinology of cows with pyometra and retained fetal membranes.

In an effort to clarify the pathogenesis of pyometra, 20 cows with retained fetal membranes and 20 without, but with contemporary calving dates were studied. They were palpated and their uteri were subjected to sample collections for bacteriologic cultural examinations twice weekly for 4 weeks. Blood samples were obtained each day and evaluated for serum progesterone concentration. Three cows without and 3 with retained fetal membranes developed pyometra during the study, resulting in 3 groups designated control (CON), cows with retained fetal membranes (RFM), and cows with pyometra (PYO). Bacterial isolations occurred less frequently in the CON group than in the PYO and RFM groups. Growth patterns of bacteria also varied between groups. Coliform and incidental bacteria disappeared from the uterus of the PYO group by the end of the 3rd week. In contrast, heavy growth of Corynebacterium pyogenes and gram-negative anaerobic bacteria developed during this same period in the PYO group. In cows with pyometra, the significant persistent pathogenic bacteria recovered were C pyogenes and gram-negative anaerobic bacteria, especially Fusobacterium necrophorum and Bacteroides melaninogenicus. Anaerobic bacteria were isolated simultaneously with C pyogenes in most cows of the PYO group, but less often in CON and RFM groups, and highest growth levels were present near the time of ovulation. Clinically, pyometra usually developed about 10 days after observation of concurrent ovulation and high growth levels of C pyogenes and gram-negative anaerobic bacteria. A hypothesis is presented for development of pyometra in the cow.

Pathologic changes and microorganisms found in bighorn sheep during a stress-related die-off.

An all-age die-off of Rocky Mountain bighorn sheep (Ovis c. canadensis Shaw) occurred from late October 1980 through March 1981 in Waterton Canyon, Colorado, with a loss of 75 to 85% of the sheep. The cause of death was a subacute to chronic bronchopneumonia and the primary etiologic agents isolated from the respiratory system were a Pasteurella sp., P. multocida, Corynebacterium pyogenes, and Protostrongylus stilesi Dikmans, 1931. The underlying predisposing factors that initiated this die-off were believed to be related to multiple chronic environmental stressors associated with the building of a dam which included human contact, vehicular traffic, atmospheric dust, noise and harassment. The die-off was succeeded by a 100% lamb mortality the following summer and a 67% lamb mortality the next two summers. The pneumonia found in these lambs was similar to that found in adult sheep during the previous die-off, except that mature lungworms were absent.