Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor,a Genus of Plant Biomass-Degrading Thermophilic Bacteria

Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C₅ and C₆ sugars primarily to hydrogen gas, lactate, acetate, and CO₂ and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.     

Enzymatic accessibility of xylans in lignocellulosic materials

The hydrolysis of fibre-bound and isolated xylans from both birch and pine wood and kraft pulps was studied using purified xylanolytic enzymes of Trichoderma reesei. Despite high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 20% of the theoretical value, apparently due to limited accessibility of the substrates. The fibre-bound xylans were as equally accessible in softwood as in hardwood pulps. The isolated xylans of wood and kraft pulps could be solubilized more extensively, with a hydrolysis yield of 50–65%. The substitution degree of the isolated xylan substrates was reflected in the different hydrolysis yields obtained by the two xylanases, with isoelectric point (pI) values of 9.0 and 5.5. On the more substituted substrates, i.e. pine kraft xylan and pine wood xylan, the two enzymes acted almost similarly, whereas on the less substituted xylan substrates, such as isolated birch kraft xylan, the pI-9.0 enzyme was more efficient. The side-group-cleaving enzymes increased only moderately the solubilization of the substrates.Correspondence to: L. Viikari     

Purification and enzymatic characterization of two β-endoxylanases from Trichoderma sp. K9301 and their actions in xylooligosaccharide production

Trichoderma sp. K9301 secreting endoxylanases with an activity of 2836 U/g (dry weight) was screened for XOs production. Two acidic β-endoxylanases EX1 (30.1 kDa) and EX2 (20.1 kDa) were purified from crude extract of the strain K9301 in solid fermentation. Action modes of EX1 and EX2 towards XOs showed similar hydrolysis characters to endoxylanases belonging to glycosyl hydrolase family 10 and 11, respectively. EX1 exhibited better affinity but lower hydrolytic efficiency than EX2 to xylans from beechwood, birchwood, and oat-spelt. They had synergistic action on xylan hydrolysis. The optimum condition to prepare XOs from corncobs was obtained as 10 mg/ml corncob xylan incubated with 10 U/mg crude enzymes at 50 °C for 3 h. The yield of XOs reached 43.3%, and only a little amount of xylose (3.1%) was simultaneously produced, suggesting the good potential of strain K9301 in XOs production.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> sp. A59 GH10 and GH11 Extracellular Endoxylanases: Application in Biomass Bioconversion

The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.     

Mutational Analysis of Endoxylanases XylA and XylB from the Phytopathogen Fusarium graminearum Reveals Comprehensive Insights into Their Inhibitor Insensitivity

Endo-β-1,4-xylanases (EC 3.2.1.8; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the “thumb” structural region. While XylB residues Cys¹⁴¹, Asp¹⁴⁸, and Cys¹⁴⁹ were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val¹⁵¹. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases.     

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.     

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.     

Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants

The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected.Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases.No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).     

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley β-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/β-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages.     

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K_a) values of 5.2 × 10⁶ to 44 × 10⁶ M⁻¹ among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.     

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.     

Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties

Xylan has a main chain consisting of β-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the − 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the − 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.

     

Engineering of a multifunctional hemicellulase

To engineer a multifunctional xylan-degrading enzyme, a chimera was created by fusing the xylanase domain of the Clostridium thermocellum xylanase (xynZ) and a dual functional arabinofuranosidase/xylosidase (DeAFc; from a compost starter mixture) through a flexible peptide linker. The xylanase domain of xynZ possesses previously unreported endoglucanase activity. The chimera, possessing the activities of xylanase, endoglucanase, arabinofuranosidase and xylosidase, was expressed in E. coli and purified. The chimera closely resembled the parental enzymes in pH, temperature optima and kinetics, and was more active than the parental enzyme mixture in the hydrolysis of natural xylans and corn stover. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Xylanolytic activities ofSpirochaeta thermophila

Spirochetes capable of degrading xylan or cellulose have not been commonly isolated, nor have their polysaccharolytic activities been characterized.Spirochaeta thermophila strain RI 19.B1 is xylanolytic and grows well at 65°C with oatspelt (OX), birchwood (BX), corncob (CCX-A) xylans, or glucuronoxylan (MGX) as the energy source. All xylans were extensively degraded and utilized during growth. About 72–82% of the initial hexuronic acids and 57–79% of initial pentoses disappeared during growth.S. thermophila possessed xylanase, xylosidase, and arabinofuranosidase enzyme activities. Low levels of these activities were detected with growth on glucose, but high expression of these activities occurred during growth on OX. All three activities were cell-associated and were more stable in cells than cell extracts. Xylan-degrading activities were measured with cells or cell extracts exposed (60 min) to a variety of temperatures (65°–85°C) and pHs (5.0–8.0). More than 50% loss of activities occurred at temperatures above 75°C. Although pH stability was affected by buffer, the optimal range was pH 6.5–7.5. These temperature and pH profiles for xylan-degrading activities coincide with those found for the growth ofS. thermophila.     

Cell Wall Metabolism in Ripening Fruit : III. Purification of an Endo-beta-1,4-xylanase that Degrades a Structural Polysaccharide of Pear Fruit Cell Walls

A β-1,4-xylanase has been purified from the mixture of carbohydrate-degrading enzymes found in a commercial preparation from cultures of Trichoderma viride. Purification from the desalted enzyme mixture is accomplished either by preparative isoelectric focusing or in two-column chromatographic steps. The xylanase has maximal activity at pH 5.0 and a molecular weight of approximately 13,000 daltons. The enzyme loses activity when heated to above 45°C. The xylanase degrades xylans from larch and pear cell walls in an apparently endo-fashion.     

β-xylosidases and a nonspecific wall-bound β-glucosidase of the yeast cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular β-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-β-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl β-D-xylopyranoside which are the best inducers of extracellular β-xylanase (EC 3.2.1.8). Various alkyl-, alkyl-1-thio- and aryl β-D-xylopyranosides were excellent of a different β-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular β-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that β-xylosidase activity in the cell walls is not due to the presence of a specific aryl β-xylosidase, but is exhibited by a nonspecific β-glucosidase (EC 3.2.1.21) inducible by β-D-xylopyranosides. The ratio of β-glucosidase and β-xylosidase activity in the cells and isolated cell walls from yeast induced by various β-xylopyranosides and β-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of β-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

Predicting the most appropriate wood biomass for selected industrial applications: comparison of wood,pulping, and enzymatic treatments using fluorescent-tagged carbohydrate-binding modules

Background

Lignocellulosic biomass will progressively become the main source of carbon for a number of products as the Earth’s oil reservoirs disappear. Technology for conversion of wood fiber into bioproducts (wood biorefining) continues to flourish, and access to reliable methods for monitoring modification of such fibers is becoming an important issue. Recently, we developed a simple, rapid approach for detecting four different types of polymer on the surface of wood fibers. Named fluorescent-tagged carbohydrate-binding module (FTCM), this method is based on the fluorescence signal from carbohydrate-binding modules-based probes designed to recognize specific polymers such as crystalline cellulose, amorphous cellulose, xylan, and mannan.

Results

Here we used FTCM to characterize pulps made from softwood and hardwood that were prepared using Kraft or chemical-thermo-mechanical pulping. Comparison of chemical analysis (NREL protocol) and FTCM revealed that FTCM results were consistent with chemical analysis of the hemicellulose composition of both hardwood and softwood samples. Kraft pulping increased the difference between softwood and hardwood surface mannans, and increased xylan exposure. This suggests that Kraft pulping leads to exposure of xylan after removal of both lignin and mannan. Impact of enzyme cocktails from Trichoderma reesei (Celluclast 1.5L) and from Aspergillus sp. (Carezyme 1000L) was investigated by analysis of hydrolyzed sugars and by FTCM. Both enzymes preparations released cellobiose and glucose from pulps, with the cocktail from Trichoderma being the most efficient. Enzymatic treatments were not as effective at converting chemical-thermomechanical pulps to simple sugars, regardless of wood type. FTCM revealed that amorphous cellulose was the primary target of either enzyme preparation, which resulted in a higher proportion of crystalline cellulose on the surface after enzymatic treatment. FTCM confirmed that enzymes from Aspergillus had little impact on exposed hemicelluloses, but that enzymes from the more aggressive Trichoderma cocktail reduced hemicelluloses at the surface.

Conclusions

Overall, this study indicates that treatment with enzymes from Trichoderma is appropriate for generating crystalline cellulose at fiber surface. Applications such as nanocellulose or composites requiring chemical resistance would benefit from this enzymatic treatment. The milder enzyme mixture from Aspergillus allowed for removal of amorphous cellulose while preserving hemicelluloses at fiber surface, which makes this treatment appropriate for new paper products where surface chemical responsiveness is required.

     

Construction of a Xylan-Fermenting Yeast Strain through Codisplay of Xylanolytic Enzymes on the Surface of Xylose-Utilizing Saccharomyces cerevisiae Cells

Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on α-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and β-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.     

Combining Substrate Specificity Analysis with Support Vector Classifiers Reveals Feruloyl Esterase as a Phylogenetically Informative Protein Group

Background

Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able to hydrolyze the ester bonds linking ferulic acid to plant cell wall polysaccharides. The diversity of substrate specificities found in the FAE family shows that this family is old enough to have experienced the emergence and loss of many activities.

Methodology/Principal Findings

In this study we evaluate the relative activity of FAEs against a variety of model substrates as a novel predictive tool for Ascomycota taxonomic classification. Our approach consists of two analytical steps; (1) an initial unsupervised analysis to cluster the FAEs substrate specificity data which were generated by cultivation of 34 Ascomycota strains and then an analysis of the produced enzyme cocktail against 10 substituted cinnamate and phenylalkanoate methyl esters, (2) a second, supervised analysis for training a predictor built on these substrate activities. By applying both linear and non-linear models we were able to correctly predict the taxonomic Class (∼86% correct classification), Order (∼88% correct classification) and Family (∼88% correct classification) that the 34 Ascomycota belong to, using the activity profiles of the FAEs.

Conclusion/Significance

The good correlation with the FAEs substrate specificities that we have defined via our phylogenetic analysis not only suggests that FAEs are phylogenetically informative proteins but it is also a considerable step towards improved FAEs functional prediction.