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

<p>Abstract</p> <p>Background</p> <p>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 <it>Trichoderma reesei </it>on xylan solubilization and enzymatic hydrolysis of cellulose.</p> <p>Results</p> <p>The solubilization of xylan in pretreated wheat straw and giant reed (<it>Arundo donax</it>) 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.</p> <p>Conclusions</p> <p>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.</p

Liquefaction of hydrothermally pretreated wheat straw at high-solids content by purified Trichoderma enzymes

Peer reviewe

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

Peer reviewe

Thermostable recombinant xylanases from Nonomuraea flexuosa and Thermoascus aurantiacus show distinct properties in the hydrolysis of xylans and pretreated wheat straw

<p>Abstract</p> <p>Background</p> <p>In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11 (GH10 and GH11, respectively) xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates.</p> <p>Results</p> <p>The xylanases from <it>Nonomuraea flexuosa </it>(Nf Xyn11A) and from <it>Thermoascus aurantiacus </it>(Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively.</p> <p>Conclusions</p> <p>Because of its high catalytic activity and good thermostability, <it>T. aurantiacus </it>xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas <it>N. flexuosa </it>xylanase shows more significant potential for the production of XOSs.</p

Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

<p>Abstract</p> <p>Background</p> <p>Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases), resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties) connected with high initial solid loadings in the lignocellulose to ethanol process.</p> <p>Results</p> <p>The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A.</p> <p>Conclusions</p> <p>Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter) hydrothermally pretreated wheat straw. Based on temperature profiling studies, <it>Thermoascus aurantiacus </it>EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.</p

Recovering Homography from Camera Captured Documents using Convolutional Neural Networks

Removing perspective distortion from hand held camera captured document images is one of the primitive tasks in document analysis, but unfortunately, no such method exists that can reliably remove the perspective distortion from document images automatically. In this paper, we propose a convolutional neural network based method for recovering homography from hand-held camera captured documents. Our proposed method works independent of document's underlying content and is trained end-to-end in a fully automatic way. Specifically, this paper makes following three contributions: Firstly, we introduce a large scale synthetic dataset for recovering homography from documents images captured under different geometric and photometric transformations; secondly, we show that a generic convolutional neural network based architecture can be successfully used for regressing the corners positions of documents captured under wild settings; thirdly, we show that L1 loss can be reliably used for corners regression. Our proposed method gives state-of-the-art performance on the tested datasets, and has potential to become an integral part of document analysis pipeline.Comment: 10 pages, 8 figure

Functionality and economic feasibility of enzymatically hydrolyzed waste bread as a sugar replacer in wheat bread making

Food-grade enzymes (alpha-amylase, amyloglucosidase, maltogenic amylase, and protease) were investigated for recycling waste bread back to wheat bread-making process. Waste bread was efficiently hydrolyzed into sugars (up to 93% glucose yield) and the best combination of enzymes was alpha-amylase (0.05 g/kg bread) and amyloglucosidase (2.5 g/kg bread). Selected enzyme hydrolysis processes were tested in wheat bread making as a (a) hydrolyzed slurry, that is hydrolyzed waste bread without solid/liquid separation and (b) syrup, that is liquid supernatant after centrifugation of the hydrolyzed waste bread. Both hydrolyzed bread slurry and syrup were successfully utilized to replace sucrose (2 and 4%) in bread making without affecting the bread quality when compared to the control bread. Techno-economic assessment revealed that this approach is 12% more economical than the current mean to dispose the bakery waste. This recycling concept showed both technical and economic potential for bakery industries to overcome their excess bread production. Novelty impact statement A new recycling process was developed by using enzymes to efficiently hydrolyze surplus bread into sugars. The use of those sugar-rich slurries and syrups in bread rework did not affect the bread quality when compared to the control bread. The recycling concept was more economical than current means to dispose waste bread, revealing the technical and economic potential for bakery industries to overcome their excess bread production.Peer reviewe

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.Peer reviewe

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>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 <it>Saccharomyces cerevisiae </it>for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.</p> <p>Results</p> <p>We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by <it>S. cerevisiae</it>. 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 <it>cbh1 </it>and <it>cbh2 </it>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 <it>S. cerevisiae </it>strains with the addition of beta-glucosidase.</p> <p>Conclusions</p> <p>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.</p