Enzyme-treated chicory for cosmetics: application assessment and techno-economic analysis.

Chicory (Cichorium intybus L.) is an important industrial crop that produces large quantities of the dietary fiber inulin in its roots. Following inulin extraction, the bagasse is typically used as animal feed, but it contains numerous bioactive secondary metabolites with potential applications in healthcare and cosmetic products. Here we assessed the antimicrobial properties of chicory biomass pre-treated with various enzymes alone and in combination to release the bioactive compounds and increase their bioavailability. We found that pre-treatment significantly increased the antimicrobial activity of this industrial by-product, yielding an extract that inhibited typical skin pathogens in a cosmetic formula challenge test. We also evaluated the valorization of chicory biomass as a bioactive cosmetic ingredient. Economic feasibility was estimated by combining our experimental results with a conceptual techno-economic analysis. Our results suggest that chicory biomass can be utilized for the sustainable production of efficacious cosmetic ingredients.

Fibrolytic enzyme treatment prior to ensiling increased press-juice and crude protein yield from grass silage.

Grass is a versatile raw material for green biorefineries and preserving it as silage provides a year-round feedstock. The objective of the current study was to evaluate the effect of fibrolytic enzyme application on silage as a feedstock for a biorefinery. Two batches of grass (mixture of timothy and meadow fescue) silages were ensiled in pilot scale after fibrolytic enzyme was applied to them at four levels. Enzyme application increased fibre degradation linearly during ensiling and increased lactic and acetic acid concentrations in the silage. Simultaneously, silage fermentation quality improved as indicated by decreasing pH and ammonia values. Press-juice and crude protein yields increased in response to the fibrolytic enzyme application, which is beneficial in a biorefinery concept for retrieving valuable nutrients from grass matrix. Optimized ensiling methodology can be considered as a pretreatment for a biorefinery process.

Enzymatic degradation of sulfite-pulped softwoods and the role of LPMOs.

BACKGROUND: Recent advances in the development of enzyme cocktails for degradation of lignocellulosic biomass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BALI™ process, yield substrates that are relatively easy to degrade. We have assessed how process conditions affect the efficiency of modern cellulase preparations in processing of such substrates. RESULTS: We show that efficient degradation of sulfite-pulped softwoods with modern, LPMO-containing cellulase preparations requires the use of conditions that promote LPMO activity, notably the presence of molecular oxygen and sufficient reducing power. Under LPMO activity-promoting conditions, glucan conversion after 48-h incubation with Cellic® CTec3 reached 73.7 and 84.3% for Norway spruce and loblolly pine, respectively, at an enzyme loading of 8 mg/g of glucan. The presence of free sulfite ions had a negative effect on hydrolysis efficiency. Lignosulfonates, produced from lignin during sulfite pretreatment, showed a potential to activate LPMOs. Spiking of Celluclast®, a cellulase cocktail with low LPMO activity, with monocomponent cellulases or an LPMO showed that the addition of the LPMO was clearly more beneficial than the addition of any classical cellulase. Addition of the LPMO in reactions with spruce increased the saccharification yield from approximately 60% to the levels obtained with Cellic® CTec3. CONCLUSIONS: In this study, we have demonstrated the importance of LPMOs for efficient enzymatic degradation of sulfite-pulped softwood. We have also shown that to exploit the full potential of LPMO-rich cellulase preparations, conditions promoting LPMO activity, in particular the presence of oxygen and reducing equivalents are necessary, as is removal of residual sulfite from the pretreatment step. The use of lignosulfonates as reductants may reduce the costs related to the addition of small molecule reductants in sulfite pretreatment-based biorefineries.

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.

Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries.

BACKGROUND: During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. RESULTS: Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous ß-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. CONCLUSIONS: In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

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.

Charge-based engineering of hydrophobin HFBI: effect on interfacial assembly and interactions.

Hydrophobins are extracellular proteins produced by filamentous fungi. They show a variety of functions at interfaces that help fungi to adapt to their environment by, for example, adhesion, formation of coatings, and lowering the surface tension of water. Hydrophobins fold into a globular structure and have a distinct hydrophobic patch on their surface that makes these proteins amphiphilic. Their amphiphilicity implies interfacial assembly, but observations indicate that intermolecular interactions also contribute to their functional properties. Here, we used the class II hydrophobin HFBI from Trichoderma reesei as a model to understand the structural basis for the function of hydrophobins. Four different variants were made in which charged residues were mutated. The residues were chosen to probe the role of different regions of the hydrophilic part of the proteins. Effects of the mutations were studied by analyzing the formation and structure of self-assembled layers, multimerization in solution, surface adhesion, binding of secondary layers of proteins on hydrophobins, and the viscoelastic behavior of the air-water interface during formation of protein films; the comparison showed clear differences between variants only in the last two analyses. Surface viscoelasticity behavior suggests that the formation of surface layers is regulated by specific interactions that lead to docking of proteins to each other. One set of mutations led to assemblies with a remarkably high elasticity at the air-water interface (1.44 N/m). The variation of binding of secondary layers of protein on surface-adsorbed hydrophobins suggest a mechanism for a proposed function of hydrophobins, namely, that hydrophobins can act as a specific adhesive layer for the binding of macromolecules to interfaces.

Effects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions.

BACKGROUND: Sugar cane internodes can be divided diagonally into four fractions, of which the two innermost ones are the least recalcitrant pith and the moderately accessible pith-rind interface. These fractions differ in enzymatic hydrolyzability due to structural differences. In general, cellulose hydrolysis in plants is hindered by its physical interaction with hemicellulose and lignin. Lignin is believed to be linked covalently to hemicellulose through hydroxycinnamic acids, forming a compact matrix around the polysaccharides. Acetyl xylan esterase and three feruloyl esterases were evaluated for their potential to fragment the lignocellulosic network in sugar cane and to indirectly increase the accessibility of cellulose. RESULTS: The hydrolyzability of the pith and pith-rind interface fractions of a low-lignin-containing sugar cane clone (H58) was compared to that of a reference cultivar (RC). Acetyl xylan esterase enhanced the rate and overall yield of cellulose and xylan hydrolysis in all four substrates. Of the three feruloyl esterases tested, only TsFaeC was capable of releasing p-coumaric acid, while AnFaeA and NcFaeD released ferulic acid from both the pith and interface fractions. Ferulic acid release was higher from the less recalcitrant clone (H58)/fraction (pith), whereas more p-coumaric acid was released from the clone (RC)/fraction (interface) with a higher lignin content. In addition, a compositional analysis of the four fractions revealed that p-coumaroyl content correlated with lignin, while feruloyl content correlated with arabinose content, suggesting different esterification patterns of these two hydroxycinnamic acids. Despite the extensive release of phenolic acids, feruloyl esterases only moderately promoted enzyme access to cellulose or xylan. CONCLUSIONS: Acetyl xylan esterase TrAXE was more efficient in enhancing the overall saccharification of sugar cane, compared to the feruloyl esterases AnFaeA, TsFaeC, and NcFaeD. The hydroxycinnamic acid composition of sugar cane fractions and the hydrolysis data together suggest that feruloyl groups are more likely to decorate xylan, while p-coumaroyl groups are rather linked to lignin. The three different feruloyl esterases had distinct product profiles on non-pretreated sugar cane substrate, indicating that sugar cane pith could function as a possible natural substrate for feruloyl esterase activity measurements. Hydrolysis data suggest that TsFaeC was able to release p-coumaroyl groups esterifying lignin.

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.

The role of endoglucanase and endoxylanase in liquefaction of hydrothermally pretreated wheat straw.

The role of endocellulases and endoxylanase during liquefaction and saccharification of hydrothermally pretreated wheat straw was studied. The use of a flow-loop setup with in-line magnetic resonance imaging enabled frequent measurements of viscosity at 55°C during saccharification at 6% total solids content. Viscosity data were complemented with off-line measurements of fiber lengths and release of soluble sugars. A clear correlation between fiber attrition and a decrease in viscosity was found. Fiber lengths and viscosity dropped quickly within the first hour and then stagnated, while sugar yields increased substantially thereafter, illustrating that liquefaction and saccharification are separate mechanisms. Both endoglucanase and endoxylanase were shown to have a significant effect on viscosity during liquefaction while the addition of endoxylanase also increased sugar yield.

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.

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.

Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs.

BACKGROUND: A vast number of organisms are known to produce structurally diversified cellulases capable of degrading cellulose, the most abundant biopolymer on earth. The generally accepted paradigm is that the carbohydrate-binding modules (CBMs) of cellulases are required for efficient saccharification of insoluble substrates. Based on sequence data, surprisingly more than 60% of the cellulases identified lack carbohydrate-binding modules or alternative protein structures linked to cellulases (dockerins). This finding poses the question about the role of the CBMs: why would most cellulases lack CBMs, if they are necessary for the efficient hydrolysis of cellulose? RESULTS: The advantage of CBMs, which increase the affinity of cellulases to substrates, was found to be diminished by reducing the amount of water in the hydrolytic system, which increases the probability of enzyme-substrate interaction. At low substrate concentration (1% w/w), CBMs were found to be more important in the catalytic performance of the cellobiohydrolases TrCel7A and TrCel6A of Trichoderma reesei as compared to that of the endoglucanases TrCel5A and TrCel7B. Increasing the substrate concentration while maintaining the enzyme-to-substrate ratio enhanced adsorption of TrCel7A, independent of the presence of the CBM. At 20% (w/w) substrate concentration, the hydrolytic performance of cellulases without CBMs caught up with that of cellulases with CBMs. This phenomenon was more noticeable on the lignin-containing pretreated wheat straw as compared to the cellulosic Avicel, presumably due to unproductive adsorption of enzymes to lignin. CONCLUSIONS: Here we propose that the water content in the natural environments of carbohydrate-degrading organisms might have led to the evolution of various substrate-binding structures. In addition, some well recognized problems of economical saccharification such as unproductive binding of cellulases, which reduces the hydrolysis rate and prevents recycling of enzymes, could be partially overcome by omitting CBMs. This finding could help solve bottlenecks of enzymatic hydrolysis of lignocelluloses and speed up commercialization of second generation bioethanol.

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.

Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity.

A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA(3)Xyl(3)), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-ß-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-ß-1,4-glucanase EG I. XYN IV was able to degrade several different ß-1,4-xylans, but was inactive on ß-1,4-mannans and ß-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble ß-1,3-ß-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward ß-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-D-glucuronic acid (MeGlcA) substituents as substrate specificity determinants.

Reed canary grass as a feedstock for 2nd generation bioethanol production.

The enzymatic hydrolysis and fermentation of reed canary grass, harvested in the spring or autumn, and barley straw were studied. Steam pretreated materials were efficiently hydrolysed by commercial enzymes with a dosage of 10-20FPU/g d.m. Reed canary grass harvested in the spring was hydrolysed more efficiently than the autumn-harvested reed canary grass. Additional ß-glucosidase improved the release of glucose and xylose during the hydrolysis reaction. The hydrolysis rate and level of reed canary grass with a commercial Trichoderma reesei cellulase could be improved by supplementation of purified enzymes. The addition of CBH II improved the hydrolysis level by 10% in 48hours' hydrolysis. Efficient mixing was shown to be important for hydrolysis already at 10% dry matter consistency. The highest ethanol concentration (20g/l) and yield (82%) was obtained with reed canary grass at 10% d.m. consistency.

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.

The role of acetyl xylan esterase in the solubilization of xylan and enzymatic hydrolysis of wheat straw and giant reed.

BACKGROUND: Due to the complexity of lignocellulosic materials, a complete enzymatic hydrolysis into fermentable sugars requires a variety of cellulolytic and xylanolytic enzymes. Addition of xylanases has been shown to significantly improve the performance of cellulases and to increase cellulose hydrolysis by solubilizing xylans in lignocellulosic materials. The goal of this work was to investigate the effect of acetyl xylan esterase (AXE) originating from Trichoderma reesei on xylan solubilization and enzymatic hydrolysis of cellulose. RESULTS: The solubilization of xylan in pretreated wheat straw and giant reed (Arundo donax) by xylanolytic enzymes and the impact of the sequential or simultaneous solubilization of xylan on the hydrolysis of cellulose by purified enzymes were investigated. The results showed that the removal of acetyl groups in xylan by AXE increased the accessibility of xylan to xylanase and improved the hydrolysis of xylan in pretreated wheat straw and giant reed. Solubilization of xylan led to an increased accessibility of cellulose to cellulases and thereby increased the hydrolysis extent of cellulose. A clear synergistic effect between cellulases and xylanolytic enzymes was observed. The highest hydrolysis yield of cellulose was obtained with a simultaneous use of cellulases, xylanase and AXE, indicating the presence of acetylated xylan within the cellulose matrix. Acetylated xylobiose and acetylated xylotriose were produced from xylan without AXE, as confirmed by atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry. CONCLUSIONS: The results in this paper demonstrate that supplementation of xylanase with AXE enhances the solubilization of xylan to some extent and, consequently, increases the subsequent hydrolysis of cellulose. The highest hydrolysis yield was, however, obtained by simultaneous hydrolysis of xylan and cellulose, indicating a layered structure of cellulose and xylan chains in the cell wall substrate. AXE has an important role in the hydrolysis of lignocellulosic materials containing acetylated xylan.

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae.

BACKGROUND: The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. RESULTS: We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. CONCLUSIONS: Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

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.