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.

Small-angle X-ray scattering and structural modeling of full-length: Cellobiohydrolase I from Trichoderma harzianum

Cellobiohydrolases hydrolyze cellulose releasing cellobiose units. They are very important for a number of biotechnological applications, such as, for example, production of cellulosic ethanol and cotton fiber processing. The Trichoderma cellobiohydrolase I (CBH1 or Cel7A) is an industrially important exocellulase. It exhibits a typical two domain architecture, with a small C-terminal cellulose-binding domain and a large N-terminal catalytic core domain, connected by an O-glycosylated linker peptide. The mechanism by which the linker mediates the concerted action of the two domains remains a conundrum. Here, we probe the protein shape and domain organization of the CBH1 of Trichoderma harzianum (ThCel7A) by small angle X-ray scattering (SAXS) and structural modeling. Our SAXS data shows that ThCel7A linker is partially-extended in solution. Structural modeling suggests that this linker conformation is stabilized by inter- and intra-molecular interactions involving the linker peptide and its O-glycosylations. © 2013 Springer Science+Business Media Dordrecht.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

Purification and Biochemical Properties of a Glucose-Stimulated beta-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Effect of glycosylation on the biochemical properties of beta-xylosidases from Aspergillus versicolor

Aspergillus versicolor grown on xylan or xylose produces two beta-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these beta-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R(f). Temperature optimum of xylan-induced and xylose-induced beta-xylosidases was 45A degrees C and 40A degrees C, respectively, and 35A degrees C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55A degrees C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.

Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

Background: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra-and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. Results: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U. mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2: 1 or 4: 1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber. Conclusion: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.

Enzyme Optimization for Lignocellulose Hydrolysis using Mechanistic Modeling

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) from Trichoderma reesei; β-glucosidase (BG), and β-xylosidase (BX) from Aspergillus niger. The model employs the synergistic action of these enzymes to predict optimum enzyme concentrations for hydrolysis of Avicel and ammonia fiber explosion (AFEX) pretreated corn stover. Glucan, glucan + xylan, glucose and glucose + xylose conversion predictions are given over a range of mass fractions of enzymes, and a range of enzyme loadings. Simulation results are compared with optimizations using statistically designed experiments. BG and BX are modeled in solution at later time points to predict the effect on glucose conversion and xylose conversion.

Effect of pH and temperature on the global compactness, structure, and activity of cellobiohydrolase Cel7A from Trichoderma harzianum

Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum (T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A from T. harzianum (Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity of Th Cel7A at different pH and temperature conditions.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Nebivolol Reduces Central Blood Pressure In Stage I Hypertensive Patients: Experimental Single Cohort Study.

Assessment of central blood pressure (BP) has grown substantially over recent years because evidence has shown that central BP is more relevant to cardiovascular outcomes than peripheral BP. Thus, different classes of antihypertensive drugs have different effects on central BP despite similar reductions in brachial BP. The aim of this study was to investigate the effect of nebivolol, a β-blocker with vasodilator properties, on the biochemical and hemodynamic parameters of hypertensive patients. Experimental single cohort study conducted in the outpatient clinic of a university hospital. Twenty-six patients were recruited. All of them underwent biochemical and hemodynamic evaluation (BP, heart rate (HR), central BP and augmentation index) before and after 3 months of using nebivolol. 88.5% of the patients were male; their mean age was 49.7 ± 9.3 years and most of them were overweight (29.6 ± 3.1 kg/m2) with large abdominal waist (102.1 ± 7.2 cm). There were significant decreases in peripheral systolic BP (P = 0.0020), diastolic BP (P = 0.0049), HR (P < 0.0001) and central BP (129.9 ± 12.3 versus 122.3 ± 10.3 mmHg; P = 0.0083) after treatment, in comparison with the baseline values. There was no statistical difference in the augmentation index or in the biochemical parameters, from before to after the treatment. Nebivolol use seems to be associated with significant reduction of central BP in stage I hypertensive patients, in addition to reductions in brachial systolic and diastolic BP.

Predictive Value Of Phase I Trials For Safety In Later Trials And Final Approved Dose: Analysis Of 61 Approved Cancer Drugs.

Phase I trials use a small number of patients to define a maximum tolerated dose (MTD) and the safety of new agents. We compared data from phase I and registration trials to determine whether early trials predicted later safety and final dose. We searched the U.S. Food and Drug Administration (FDA) website for drugs approved in nonpediatric cancers (January 1990-October 2012). The recommended phase II dose (R2PD) and toxicities from phase I were compared with doses and safety in later trials. In 62 of 85 (73%) matched trials, the dose from the later trial was within 20% of the RP2D. In a multivariable analysis, phase I trials of targeted agents were less predictive of the final approved dose (OR, 0.2 for adopting ± 20% of the RP2D for targeted vs. other classes; P = 0.025). Of the 530 clinically relevant toxicities in later trials, 70% (n = 374) were described in phase I. A significant relationship (P = 0.0032) between increasing the number of patients in phase I (up to 60) and the ability to describe future clinically relevant toxicities was observed. Among 28,505 patients in later trials, the death rate that was related to drug was 1.41%. In conclusion, dosing based on phase I trials was associated with a low toxicity-related death rate in later trials. The ability to predict relevant toxicities correlates with the number of patients on the initial phase I trial. The final dose approved was within 20% of the RP2D in 73% of assessed trials.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.