Exopolysaccharide production by a marine cyanobacterium Cyanothece sp.

The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBHI. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.     

Isotherms for adsorption of cellobiohydrolase I and II fromtrichoderma reesei on microcrystalline cellulose

Adsorption to microcrystalline cellulose (Avicel) of pure cellobiohydrolase I and II (CBH I and CBH II) fromTrichoderma reesei has been studied. Adsorption isotherms of the enzymes were measured at 4‡C using CBH I and CBH II alone and in reconstituted equimolar mixtures. Several models (Langmuir, Freundlich, Temkin, Jovanovic) were tested to describe the experimental adsorption isotherms. The isotherms did not follow the basic (one site) Langmuir equation that has often been used to describe adsorption isotherms of cellulases; correlation coefficients (R²) were only 0.926 and 0.947, for CBH I and II, respectively. The experimental isotherms were best described by a model of Langmuir type with two adsorption sites and by a combined Langmuir-Freundlich model (analogous to the Hill equation); using these models the correlation coefficients were in most cases higher than 0.995. Apparent binding parameters derived from the two sites Langmuir model indicated stronger binding of CBH II compared to CBH I; the distribution coefficients were 20.7 and 3.7 L/g for the two enzymes, respectively. The binding capacity, on the other hand, was higher for CBH I, 1.0 Μmol (67 mg) per gram Avicel, compared to 0.57 Μmol/g (30 mg/g) for CBH II. The isotherms when analyzed with the combined Langmuir-Freundlich model indicated presence of unequal binding sites on cellulose and/or negative cooperativity in the binding of the enzyme molecules.     

Rotation of Cellulose Ribbons During Degradation with Fungal Cellulase

Degradation of bacterial cellulose with a commercial cellulase, Celluclast 1.5L (Novo Nordisk), from the fungus Trichoderma reesei, causes a rotational movement of the cellulose microfibrils. Purified cellulases (CBH I, CBH II, and EG II) do not induce rotation of bacterial cellulose, however, ratios of CBH I and EG II do cause rotation of bacterial cellulose. Equimolar amounts of CBH I or CBH II and EG II do not result in motion during degradation. Based on these observations, we provide further evidence supporting, at least on theoretical grounds, the hypothesis that cellulose chains have intrinsic chirality. As the cellulase enzymes interact with and degrade the cellulose fibrils, the crystalline structure of the cellulose is altered, allowing the linear cellulose polymers to relax into a lower energy state, thus relieving the strain induced by crystallization of the nascent -glucan chains during the biogenesis of the microfibril. This conversion of crystalline bacterial ribbons into more relaxed conformations produces the rotation observed during the treatment of bacterial cellulose with cellulase.     

Trichoderma reesei cellulases and their core domains in the hydrolysis and modification of chemical pulp

The action of monocomponent Trichoderma reesei endoglucanases (EG I, EG II; EC 3.2.1.4) and cellobiohydrolases (CBH I, CBH II; EC 3.2.1.91) and their core proteins was compared using isolated celluloses and bleached chemical pulp. The presence of cellulose binding domain (CBD) in the intact enzymes did not affect their action against soluble substrates. In the case of insoluble isolated celluloses and the chemical pulp the presence of CBD enhanced the enzymatic hydrolysis of cellulose. The effect of CBD was more pronounced in the cellobiohydrolases, hydrolysing mainly crystalline cellulose, than in the endoglucanases which were more efficient in hydrolysing amorphous cellulose. The pulp properties measured, that is, viscosity and strength after PFI refining, were equally affected by the treatment with intact enzymes and corresponding core proteins, suggesting that the presence of CBD in intact cellulases affects mainly the cellulose hydrolysis level and less the mode of action of T. reesei cellulases in pulp. The better beatability of the bleached chemical pulp treated with intact endoglucanases than that treated with the corresponding core proteins suggests that the presence of CBD in endoglucanases could, however, result in beneficial effects on pulp properties.     

Horticultural Waste as the Substrate for Cellulase and Hemicellulase Production by Trichoderma reesei Under Solid-State Fermentation

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), β-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.     

Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis

The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.     

A new thermostable endoglucanase,Acidothermus cellulolyticus E1

A new thermostable endoglucanase,Acidothermus cellulolyticus E1, and another bacterial endoglucanase, E₅ fromThermomonospora fusca, each exhibit striking synergism with a fungal cellobiohydrolase (Trichoderma reesei CBH I) in the saccharification of microcrystalline cellulose. In neither case did the ratio of endoglucanase to exoglucanase that demonstrated maximum synergism coincide exactly with the ratio that actually released the maximum quantity of soluble sugar for a given total cellulase loading. The difference between the two ratios, after significant hydrolysis of the substrate, was considerably larger in the case ofA. cellulolyticus E1. For both endoglucanase pairings with CBH I, the offset between the ratio for maximum synergism and the ratio for maximal soluble sugar production was found to be a function of digestion time.     

The effect of Trichoderma reesei cellulases and hemicellulases on the paper technical properties of never-dried bleached kraft pulp

Four purified cellulases, a xylanase and mannanase from Trichoderma reesei were used to treat never-dried bleached pine kraft pulp prior to refining, and the effects on pulp properties were evaluated. The enzymatic treatments hydrolysed up to 0.8% of pulp dry weight. The results demonstrated that the individual cellulases have profoundly different modes of action in modifying pulp carbohydrates. This is especially clear when comparing their effects at the same level of hydrolysis. Pretreatment with cellobiohydrolases I (CBH I) and II (CBH II) had virtually no effect on the development of pulp properties during refining, except for a slight decrease in strength properties. On the contrary, endoglucanase I (EG I) and endoglucanase II (EG II) improved the beatability of the pulp as measured by Schopper--Riegler value, sheet density and Gurley air resistance. Of the endoglucanases, EG II was most effective in improving the beating response. The combinations of CBH I with EG I and EG II had similar effects on the pulp properties as the endoglucanases alone, although the amount of hydrolysed cellulose was increased. Pretreatments with xylanase or mannanase did not appear to modify the pulp properties. The same enzyme treatments which improved the beatability, however, slightly impaired the pulp strength, especially tear index at the enzyme dosages used. When compared at a given level of cellulose hydrolysis, the negative effect of EG II on strength properties was more pronounced compared with EG I. Thus, the exploitation of cellulases for fibre treatments requires careful optimization of both enzyme composition and dosage. Since the endoglucanases had no positive effect on the development of tensile strength, it is suggested that the explanation for the increased beating response is increased fibre breakage and formation of fines, rather than improved flexibilization. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interactions of the complete cellobiohydrolase I from Trichodera reesei with microcrystalline cellulose Iβ

We describe the construction of a model complex of the cellobiohydrolase I (CBH I) cellulase from Trichoderma reesei bound to a cellulose microfibril in an aqueous environment for use in molecular dynamics (MD) simulations. Preliminary characterization from the initial phases of an MD simulation of this complex is also described. The linker sequence between the two globular domains was found to be quite flexible, and the oligosaccharides bound to this linker were found to prefer to be splayed like the spokes in a wheel due to their hydration requirements. The overall conformations of the two globular domains remained stable in the simulations, although both underwent changes in their orientations.     

Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica

The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.     

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their superna tants after growth in a microcrystalline cellulose medium, using carboxy methylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Flow-Microcalorimetric Determination of Cellobiase Activity and Its Inhibition by Glucose

Abstract

A method for measuring cellobiase activity of the Trichoderma reesei CCF 1853 cellulase complex using a Thermal Activity Monitor and a flow - mix mode is described. The kinetic constant K_M and the linear dependence of dQ_max/dt (the maximum heat flow at the total saturation of enzyme with substrate) on the enzyme concentration were determined. The process of the end product inhibition of cellobiase activity by glucose has been observed too. The obtained results allow to determine the mechanism of the inhibition and an inhibition constant for glucose.

The procedure is completely general in nature and is applicable to other enzymatic systems.     

Mixtures of Thermostable Enzymes Show High Performance in Biomass Saccharification

Optimal enzyme mixtures of six Trichoderma reesei enzymes and five thermostable enzyme components were developed for the hydrolysis of hydrothermally pretreated wheat straw, alkaline oxidised sugar cane bagasse and steam-exploded bagasse by statistically designed experiments. Preliminary studies to narrow down the optimization parameters showed that a cellobiohydrolase/endoglucanase (CBH/EG) ratio of 4:1 or higher of thermostable enzymes gave the maximal CBH-EG synergy in the hydrolysis of hydrothermally pretreated wheat straw. The composition of optimal enzyme mixtures depended clearly on the substrate and on the enzyme system studied. The optimal enzyme mixture of thermostable enzymes was dominated by Cel7A and required a relatively high amount of xylanase, whereas with T. reesei enzymes, the high proportion of Cel7B appeared to provide the required xylanase activity. The main effect of the pretreatment method was that the required proportion of xylanase was higher and the proportion of Cel7A lower in the optimized mixture for hydrolysis of alkaline oxidised bagasse than steam-exploded bagasse. In prolonged hydrolyses, less Cel7A was generally required in the optimal mixture. Five-component mixtures of thermostable enzymes showed comparable hydrolysis yields to those of commercial enzyme mixtures.     

Hydrolysis of steam-pretreated lignocellulose

The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose. Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures. Adsorption isotherms were determined at 4 and 40 degrees C during 90-min reaction times. Both CBH I and EG II adsorbed stronger at 40 than at 4 degrees C. The time course of adsorption and hydrolysis, 3 min to 48 h, was studied at 40 degrees C. About 90% of the cellulases were adsorbed within 2 h. The hydrolysis rate was high in the beginning but decreased during the time course. Based on adsorption data, the hydrolysis and synergism were analyzed as function of adsorbed enzyme. CBH I showed a linear correlation between hydrolysis and adsorbed enzyme, whereas for EG II the corresponding curve leveled off at both 4 and 40 degrees C. At low conversion, below 1%, EG II produced as much soluble sugars as CBH I. At higher conversion, CBH I was more efficient than EG II. The synergism as function of adsorbed enzyme increased with bound enzyme before reaching a stable value of about 2. The effect of varying the ratio of CBH I:EG II was studied at fixed total enzyme loading and by changing the ratio between the enzymes. Only a small addition (5%) of EG II to a CBH I solution was shown to be sufficient for nearly maximal synergism. The ratio between EG II and CBH I was not critical. The ratio 40% EG II:60% CBH I showed similar conversion to 5% EG II:95% CBH I. Modifications of the conventional endo-exo synergism model are proposed.     

Effect of Trichoderma reesei Proteinases on the Affinity of an Inorganic-Binding Peptide

An inorganic-binding peptide sequence with high affinity to silica-containing materials was fused to a glycoside hydrolase GH26 mannanase, ManA, from the extremely thermophilic bacterium Dictyoglomus thermophilum. The resulting recombinant enzyme produced in Escherichia coli, ManA-Linker, displayed high binding affinity towards synthetic zeolite while retaining its catalytic activity at 80 °C. ManA-Linker was able to bind to the zeolite at different pH levels, indicating a true pH-independent binding. However, complete degradation of the peptide linker was observed when the recombinant ManA-Linker was exposed to the supernatant from the filamentous fungus Trichoderma reesei. This degradation was caused by extracellular proteinases produced by T. reesei during its growth phase. Several derivatives of ManA-Linker were designed and expressed in E. coli. All the derivatives carrying a single sequence of the linker were still susceptible to T. reesei proteinase degradation. Complete substitution of the linker sequence by (GGGGS)₁₆ resulted in a proteinase-resistant ManA derivative, ManA-Linker-(GGGGS)₁₆, which was able to bind to zeolite in a pH-dependent manner.     

Display of Escherichia coli Phytase on the Surface of Bacillus subtilis Spore Using CotG as an Anchor Protein

Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12 h, with maximum activity observed at pH 4.5. The highest enzyme activity seen at 55 °C and thermal stability measurements demonstrated that more than 30% activity remained after 30 min incubation at 60 °C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.

     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Cobalt activation of Bacillus BR449 thermostable nitrile hydratase expressed in Escherichia coli

Expression of nitrile hydratase enzymes utilized in a new “green” process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobaltion into this host. We describe the expression of a thermostable nitrile hydratase from a moderatethermophile Bacillus sp. BR449 in E. coli in which the cobaltrequired for enzyme activation is introduced by incubation, of the apoenzyme in the presence of Co⁺⁺ ion at 50°C, yielding active and thermostable, enzyme.