Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood.

Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these--two in glycoside hydrolase family 5, one in family 10, and one in family 12--have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.

Changes in submicrometer structure of enzymatically hydrolyzed microcrystalline cellulose.

To understand the limitations occurring during enzymatic hydrolysis of cellulosic materials in renewable energy production, we used wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), X-ray microtomography, and transmission electron microscopy (TEM) to characterize submicrometer changes in the structure of microcrystalline cellulose (Avicel) digested with the Trichoderma reesei enzyme system. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures, similar elongated and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results of rewetted samples suggested a slight change in the structure in scale of 10-20 nm, whereas the WAXS results confirmed that the degree of crystallinity and the crystal sizes remained unchanged. This indicates that the enzymes act on the surface of cellulose bundles and are unable to penetrate into the nanopores of wet cellulose.

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases.

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.

Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces.

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.

Hydrothermal treatment followed by enzymatic hydrolysis and hydrothermal carbonization as means to valorise agro- and forest-based biomass residues.

The suitability of several abundant but underutilized agro and forest based biomass residues for hydrothermal treatment followed by enzymatic hydrolysis as well as for hydrothermal carbonization was studied. The selected approaches represent simple biotechnical and thermochemical treatment routes suitable for wet biomass. Based on the results, the hydrothermal pre-treatment followed by enzymatic hydrolysis seemed to be most suitable for processing of carbohydrate rich corn leaves, corn stover, wheat straw and willow. High content of thermally stable components (i.e. lignin) and low content of ash in the biomass were advantageous for hydrothermal carbonization of grape pomace, coffee cake, Scots pine bark and willow.

Expression of Two Novel ß-Glucosidases from Chaetomium atrobrunneum in Trichoderma reesei and Characterization of the Heterologous Protein Products.

Two novel GH3 family thermostable ß-glucosidases from the filamentous fungus Chaetomium atrobrunneum (CEL3a and CEL3b) were expressed in Trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. Both enzymes were active over a wide range of pH as compared to Neurospora crassa ß-glucosidase GH3-3, which was also expressed in T. reesei and purified. The optimum temperature of both C. atrobrunneum enzymes was around 60 °C at pH 5, and both enzymes had better thermal and pH stability and higher resistance to metallic compounds and to glucose inhibition than GH3-3. They also showed higher activity against oligosaccharides composed of glucose units and linked with ß-1,4-glycosidic bonds and moreover, had higher affinity for cellotriose over cellobiose. In hydrolysis tests against Avicel cellulose and steam-exploded sugarcane bagasse, performed at 45 °C, particularly the CEL3a enzyme performed similarly to N. crassa GH3-3 ß-glucosidase. Taking into account the thermal stability of the C. atrobrunneum ß-glucosidases, they both represent promising alternatives as enzyme mixture components for improved cellulose saccharification at elevated temperatures.

Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei.

Trichoderma reesei produces five known endoglucanases. The most studied are Cel7B (EG I) and Cel5A (EG II) which are the most abundant of the endoglucanases. We have performed a characterisation of the enzymatic properties of the less well-studied endoglucanases Cel12A (EG III), Cel45A (EG V) and the catalytic core of Cel45A. For comparison, Cel5A and Cel7B were included in the study. Adsorption studies on microcrystalline cellulose (Avicel) and phosphoric acid swollen cellulose (PASC) showed that Cel5A, Cel7B, Cel45A and Cel45Acore adsorbed to these substrates. In contrast, Cel12A adsorbed weakly to both Avicel and PASC. The products formed on Avicel, PASC and carboxymethylcellulose (CMC) were analysed. Cel7B produced glucose and cellobiose from all substrates. Cel5A and Cel12A also produced cellotriose, in addition to glucose and cellobiose, on the substrates. Cel45A showed a clearly different product pattern by having cellotetraose as the main product, with practically no glucose and cellobiose formation. The kinetic constants were determined on cellotriose, cellotetraose and cellopentaose for the enzymes. Cel12A did not hydrolyse cellotriose. The k(Cat) values for Cel12A on cellotetraose and cellopentaose were significantly lower compared with Cel5A and Cel7B. Cel7B was the only endoglucanase which rapidly hydrolysed cellotriose. Cel45Acore did not show activity on any of the three studied cello-oligosaccharides. The four endoglucanases' capacity to hydrolyse beta-glucan and glucomannan were studied. Cel12A hydrolysed beta-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with beta-glucan. The capability of Cel45A to hydrolyse glucomannan was higher than that observed for Cel12A, Cel5A and Cel7B. The results indicate that Cel45A is a glucomannanase rather than a strict endoglucanase.

Regioselective deacetylation of cellulose acetates by acetyl xylan esterases of different CE-families.

Cellulose acetate (CA) was found to be a substrate of several acetyl xylan esterases (AXE). Eight AXE from different carbohydrate esterase (CE) families were tested on their activity against CA with a degree of substitution of 0.7 and 1.4. The classification of the AXEs into CE families according to their structure by hydrophobic cluster analysis followed clearly their activity against CA. Within the same CE family similar, and between the CE families different deacetylation behaviours could be observed. Furthermore, each esterase family showed a distinct regioselective mode of action. The CE 1 family enzymes regioselectively cleaved the substituents in C2- and C3-position, while CE 5 family enzymes only cleaved the acetyl groups in C2-position. CE 4 family enzymes seemed to interact only with the substituents in C3-position. Evidence was found that the deacetylation reaction of the CE 1 family enzymes proceeded faster in C2- than in C3-position of CA. The enzymes were able to cleave acetyl groups from fully substituted anhydroglucose units.

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.

Assessment of the enzymatic hydrolysis profile of cellulosic substrates based on reducing sugar release.

The activity profile of a 1:0.30 mixture of Celluclast 1.5L FG and Novozym 188 (Novozymes) was investigated using Whatman #1 filter paper (W1FP) as a single substrate for hydrolysis. The procedure was based on the ability of the enzymes to release total (RS(Tot)), insoluble (RS(Insol)) and soluble (RS(Sol)) reducing sugars from W1FP. RS(Insol) was used to estimate endoglucanase (EnG) activity whereas exoglucanases (ExG) were assessed by measuring RSSol in the presence of δ-gluconolactone. Finally, the ß-glucosidase (ßG) activity was derived from the difference between RS(Sol) measurements in the presence and absence of δ-gluconolactone. When this analytical procedure was applied to W1FP using 9.64 mg mL(-1) of the enzyme mixture, the relative contributions of EnG, ExG and ßG to the total cellulase activity were 63.28%, 12.02% and 24.70%, respectively. Also, this ratio changed with changes in the enzyme loading, giving a new insight into the synergy that exists among the enzymes.

Xylan as limiting factor in enzymatic hydrolysis of nanocellulose.

The role of xylan as a limiting factor in the enzymatic hydrolysis of cellulose was studied by hydrolysing nanocellulose samples prepared by mechanical fibrillation of birch pulp with varying xylan content. Analyzing the nanocelluloses and their hydrolysis residues with dynamic FT-IR spectroscopy revealed that a certain fraction of xylan remained tightly attached to cellulose fibrils despite partial hydrolysis of xylan with xylanase prior to pulp fibrillation and that this fraction remained in the structure during the hydrolysis of nanocellulose with cellulase mixture as well. Thus, a loosely bound fraction of xylan was predicted to have been more likely removed by purified xylanase. The presence of loosely bound xylan seemed to limit the hydrolysis of crystalline cellulose, indicated by an increase in cellulose crystallinity and by preserved crystal width measured with wide-angle X-ray scattering. Removing loosely bound xylan led to a proportional hydrolysis of xylan and cellulose with the cellulase mixture.

A novel alkaline oxidation pretreatment for spruce, birch and sugar cane bagasse.

Alkaline oxidation pretreatment was developed for spruce, birch and sugar cane bagasse. The reaction was carried out in alkaline water solution under 10 bar oxygen pressure and at mild reaction temperature of 120-140°C. Most of the lignin was solubilised by the alkaline oxidation pretreatment and an easily hydrolysable carbohydrate fraction was obtained. After 72 h hydrolysis with a 10 FPU/g enzyme dosage, glucose yields of 80%, 91%, and 97%, for spruce, birch and bagasse, respectively, were achieved. The enzyme dosage could be decreased to 4 FPU/g without a major effect in terms of the hydrolysis performance. Compared to steam explosion alkaline oxidation was found to be significantly better in the conditions tested, especially for the pretreatment of spruce. In hydrolysis and fermentation at 12% d.m. consistency an ethanol yield of 80% could be obtained with both bagasse and spruce in 1-3 days.

Hot water extraction and steam explosion as pretreatments for ethanol production from spruce bark.

Spruce bark is a source of interesting polyphenolic compounds and also a potential but little studied feedstock for sugar route biorefinery processes. Enzymatic hydrolysis and fermentation of spruce bark sugars to ethanol were studied after three different pretreatments: steam explosion (SE), hot water extraction (HWE) at 80 °C, and sequential hot water extraction and steam explosion (HWE+SE), and the recovery of different components was determined during the pretreatments. The best steam explosion conditions were 5 min at 190 °C without acid catalyst based on the efficiency of enzymatic hydrolysis of the material. However, when pectinase was included in the enzyme mixture, the hydrolysis rate and yield of HWE bark was as good as that of SE and HWE+SE barks. Ethanol was produced efficiently with the yeast Saccharomyces cerevisiae from the pretreated and hydrolysed materials suggesting the suitability of spruce bark to various lignocellulosic ethanol process concepts.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger ß-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance.

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis.

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood.

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes. The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-ß-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan. When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.

A new approach for studying correlations between the chemical structure and the rheological properties in carboxymethyl cellulose.

Two model sodium carboxymethyl celluloses (CMC) with similar monomer composition but with significant differences in the viscoelastic properties, that could not be assigned to variations in the average molar mass or molar mass distribution, were investigated with respect to the fraction of nonsubstituted cellulose segments in the polymers. The CMCs were hydrolyzed by a purified highly selective endoglucanase. The average molar mass and molar mass distribution of the enzyme products, as measured by size-exclusion chromatography with online multi-angle light scattering and refractive index detection (SEC/MALS/RI), revealed that the enzyme-catalyzed hydrolysis was more effective on one of the CMCs. To investigate whether this was due to a higher fraction of nonsubstituted cellulose segments in the polymer, the concentrations of nonsubstituted enzyme products, e.g., cellotetraose and cellopentaose, were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was concluded that the two CMCs displayed significant differences in the fraction of nonsubstituted cellulose segments. Furthermore, the CMC with the strongest attractive intermolecular interactions, according to rheometry, also contained the highest fraction of nonsubstituted cellulose segments.

Thermostable enzymes in lignocellulose hydrolysis.

Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes, enhanced stability allowing improved hydrolysis performance and increased flexibility with respect to process configurations, all leading to improvement of the overall economy of the process. New thermostable cellulase mixtures were composed of cloned fungal enzymes for hydrolysis experiments. Three thermostable cellulases, identified as the most promising enzymes in their categories (cellobiohydrolase, endoglucanase and beta-glucosidase), were cloned and produced in Trichoderma reesei and mixed to compose a novel mixture of thermostable cellulases. Thermostable xylanase was added to enzyme preparations used on substrates containing residual hemicellulose. The new optimised thermostable enzyme mixtures were evaluated in high temperature hydrolysis experiments on technical steam pretreated raw materials: spruce and corn stover. The hydrolysis temperature could be increased by about 10-15 degrees C, as compared with present commercial Trichoderma enzymes. The same degree of hydrolysis, about 90% of theoretical, measured as individual sugars, could be obtained with the thermostable enzymes at 60 degrees C as with the commercial enzymes at 45 degrees C. Clearly more efficient hydrolysis per assayed FPU unit or per amount of cellobiohydrolase I protein used was obtained. The maximum FPU activity of the novel enzyme mixture was about 25% higher at the optimum temperature at 65 degrees C, as compared with the highest activity of the commercial reference enzyme at 60 degrees C. The results provide a promising basis to produce and formulate improved enzyme products. These products can have high temperature stability in process conditions in the range of 55-60 degrees C (with present industrial products at 45-50 degrees C) and clearly improved specific activity, essentially decreasing the protein dosage required for an efficient hydrolysis of lignocellulosic substrates. New types of process configurations based on thermostable enzymes are discussed.

Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry.

An on-line system based on microdialysis sampling (MD), micro-high performance anion exchange chromatography (micro-HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on-line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5-mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro-HPAEC-IPED/MS and on-line desalting of hydrolysates enabled injection (5 microL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm-selectivity of the MD membrane (30 kDa cut-off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on-line with micro-HPAEC-IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co-elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean-up combined with on-line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.