Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

Cell wall hemicelluloses and pectins are O‐acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O‐acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody‐tissue‐specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall‐bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β‐1,4‐endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1‐expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.     

食用菌半纤维素酶系研究进展

半纤维素是一类取之不尽而又亟待开发利用的碳水化合物。对食用菌半纤维素酶系的研究已经积累了诸多资料,主要包括β-1,4-内切木聚糖酶、β-木糖苷酶、α-阿拉伯呋喃糖苷酶、乙酰木聚糖酯酶、α-葡萄糖醛酸酶的研究。主要对半纤维素酶系,尤其是木聚糖酶的生物化学与分子生物学研究进行了简要概括,包括结构、酶学性质、基因的克隆与表达等,并综述了在食用菌生产中半纤维素酶活性的变化,最后展望了食用菌半纤维素酶系今后的主要研究方向。     

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Background

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants.

Methods

Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests.

Key Results

An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3–4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass.

Conclusions

This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.     

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H₂O₂ production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H₂O₂.     

Purification and Cooperative Activity of Enzymes Constituting the Xylan-Degrading System of Thermomonospora fusca

The thermophilic actinomycete Thermomonospora fusca produced endoxylanase, α-arabinofuranosidase, β-xylosidase, and acetyl esterase activities maximally during growth on xylan. Growth yields on glucose, xylose, or arabinose were comparable, but production of endoxylanase and β-xylosidase was not induced on these substrates. The crude xylanase activity was thermostable and relatively resistant to end product inhibition by xylobiose and xylan hydrolysis products. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gels, but only a 32-kDa protein exhibiting three isomeric forms could be purified by fast protein liquid chromatography. Endoglucanases were also identified in carboxymethylcellulose-grown cultures, and their distinction from endoxylanases was confirmed. α-Arabinofuranosidase activity was due to a single dimeric protein of 92 kDa, which was particularly resistant to end product inhibition by arabinose. Three bands of acetyl esterase activity were detected by zymogram analysis, and there was evidence that these mainly consisted of an intracellular 80-kDa protein secreted to yield active 40-kDa subunits in the culture supernatant. The acetyl esterases were found to be responsible for acetyl xylan esterase activity in T. fusca, in contrast to the distinction proposed in some other systems. The addition of purified βxylosidase to endoxylanase increased the hydrolysis of xylan, probably by relieving end product inhibition. The enhanced saccharification of wheat straw caused by the addition of purified α-arabinofuranosidase to T. fusca endoxylanase suggested a truly synergistic relationship, in agreement with proposals that arabinose side groups on the xylan chain participate in cross-linking within the plant cell wall structure.     

Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation to plant cell wall degradation

Summary Trans-feruloyl and trans-p-coumaroyl esterases were found in the culture filtrates of two monocentric (Piromyces MC-1, Neocallimastix MC-2) and three polycentric (Orpinomyces PC-2, Orpinomyces PC-3, and PC-1, an unnamed genus with uniflagellated zoospores) isolates of anaerobic rumen fungi. Treatment of cell walls of Coastal bermudagrass shoots with the filtrates released the trans isomers of ferulic and p-coumaric acids; results of microscopic observations indicated that fungal isolates degraded primarily unlignified cell walls in leaf blades and stems. A greater proportion of ferulic than p-coumaric acid was released by this treatment when compared with the amounts of the acids released by saponification of the walls with 1 M NaOH. The filtrates also showed esterase activities against the trans isomers of methyl ferulate and methyl p-coumarate, with ferulic acid being released at a faster rate than p-coumaric acid. Assays for other cell-wall-degrading enzymes (xylanase, -xylosidase, -l-arabinosidase, cellulase, -glucosidase) indicated that only -xylosidase correlated with ferulate and p-coumarate esterase activities. The monocentric isolate MC-2 had the highest esterase activity against both the plant cell wall and methyl ester substrates and the highest specific activities of acetyl esterase, -xylosidase, -l-arabinosidase, cellulase and -glucosidase. Isolate MC-2 produced substantially greater amounts of feruloyl and p-coumaroyl esterase when the growth substrate contained higher levels of saponifiable ferulic and p-coumaric acids. Offprint requests to: W. S. Borneman     

AtBGAL10 is the main xyloglucan β-galactosidase in Arabidopsis, and its absence results in unusual xyloglucan subunits and growth defects

In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. In Arabidopsis (Arabidopsis thaliana), a significant proportion of xyloglucan side chains contain β-galactose linked to α-xylose at O2. In this work, we identified AtBGAL10 (At5g63810) as the gene responsible for the majority of β-galactosidase activity against xyloglucan. Xyloglucan from bgal10 insertional mutants was found to contain a large proportion of unusual subunits, such as GLG and GLLG. These subunits were not detected in a bgal10 xyl1 double mutant, deficient in both β-galactosidase and α-xylosidase. Xyloglucan from bgal10 xyl1 plants was enriched instead in XXLG/XLXG and XLLG subunits. In both cases, changes in xyloglucan composition were larger in the endoglucanase-accessible fraction. These results suggest that glycosidases acting on nonreducing ends digest large amounts of xyloglucan in wild-type plants, while plants deficient in any of these activities accumulate partly digested subunits. In both bgal10 and bgal10 xyl1, siliques and sepals were shorter, a phenotype that could be explained by an excess of nonreducing ends leading to a reinforced xyloglucan network. Additionally, AtBGAL10 expression was examined with a promoter-reporter construct. Expression was high in many cell types undergoing wall extension or remodeling, such as young stems, abscission zones, or developing vasculature, showing good correlation with α-xylosidase expression.     

Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation

Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea

Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.     

Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.     

The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family

As a component of the array of enzymes produced by micro-organisms to deconstruct plant cell walls, feruloyl esterases hydrolyze phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure, making material more accessible to glycosyl hydrolases. Here, we describe the first crystal structure of the non-modular type-A feruloyl esterase from Aspergillus niger (AnFaeA) solved at 2.5A resolution. AnFaeA displays an alpha/beta hydrolase fold similar to that found in fungal lipases and different from that reported for other feruloyl esterases. Crystallographic and site-directed mutagenesis studies allow us to identify the catalytic triad (Ser133-His247-Asp194) that forms the catalytic machinery of this enzyme. The active-site cavity is confined by a lid (residues 68-80), on the analogy of lipases, and by a loop (residues 226-244) that confers plasticity to the substrate-binding site. The lid presents a high ratio of polar residues, which in addition to a unique N-glycosylation site stabilises the lid in an open conformation, conferring the esterase character to this enzyme. A putative model for bound 5,5'-diferulic acid-linked arabinoxylan has been built, pointing to the more relevant residues involved in substrate recognition. Comparison with structurally related lipases reveals that subtle amino acid and conformational changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties, while comparison with functionally related proteins points to a functional convergence after evolutionary divergence within the feruloyl esterases family.     

Four glycosidases secreted by Collectotrichum lindemuthianum

The fungal plant pathogen, Collectotrichum lindemuthianum, was grown in culture with either galactose, arabinose or pectin as the carbon source resulting in the selective secretion of α-galactosidase, α-arabinofuranosidase and exopolygalacturonase, respectively. Each enzyme has been purified by ion exchange and gel permeation chromatography. In addition, a β-glucosidase was purified from the culture filtrate or arabinose grown fungus. The purified α-galactosidase and α-arabinosidase preparations were found to be essentially free of other carbohydrases while the β-glucosidase and exopolygalacturonase preparations contain contaminating activities.     

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification.     

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white‐rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col‐0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl‐4‐O‐methyl‐d ‐glucopyranuronate and could target ester linkages that contribute to lignin–carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf‐yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT‐IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross‐linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross‐linking within plant cell walls.     

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

转拟南芥NPR1基因桂99 T3代植株的抗病性及农艺性状表现

以转拟南芥AtNPR1基因的恢复系品种桂99T3代纯合株系为材料,考查其农艺性状及其抗病性,并比较转基因植株与桂99侵染水稻白叶枯病菌后的农艺性状。结果表明:转基因植株表现出对水稻白叶枯病的抗性显著增强77%以上;穗长、剑叶长、有效穗数、一次枝梗数、每穗实粒数、单株产量和谷粒宽等农艺性状与未转基因桂99无显著差别。在受到水稻白叶枯病菌侵染后,转基因植株的一次枝梗数、每穗粒数、每穗实粒数和单株产量等方面均比对照桂99高出13%～78%。说明AtNPR1基因增强了水稻的抗病能力,从而降低了病害引起的产量损失。转基因植株的恢复力不受影响,稻米品质比桂99更加优良。本工作为转基因水稻抗病育种的研究奠定了基础。     

Feruloyl Esterases as Biotechnological Tools： Current and Future Perspectives

Feruloyl esterases represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds between plant cell wall polysaccharide and phenolic acid. They are widely distributed in plants and microorganisms. Besides lipases, a considerable number of microbial feruloyl esterases have also been discovered and overexpressed. This review summarizes the latest research on their classification, production, and biophysicochemical properties. Special emphasis is given to the importance of that type of enzyme and their related phenolic ferulic acid compound in biotechnological processes, and industrial and medicinal applications.     

What can feruloyl esterases do for us?

The role of feruloyl esterases in plant wall development, in gut health, and in the breakdown of plant biomass for the production of bioactive phytochemicals and biofuel is covered in this review. These enzymes have potential roles in stomatal cell function and the phenolic substitutions and cross-linkages between plant cell wall components. As more plant genomes are sequenced, the role of ferulic acid and feruloyl esterases in planta may be better understood. In human and ruminal digestion, these enzymes are important to de-esterify dietary fibre, releasing hydroxycinnamates and derivatives which have been shown to have positive health effects, such as antioxidant, anti-inflammatory and anti-microbial activities. They are also involved in colonic fermentation where their extracellular and intracellular activities in the microbiota improve the breakdown of polysaccharides and increase microbial production of short chain fatty acids. Their specificity can also be employed to synthesize bioactive compounds for cosmetic and health applications. The enzymatic disassembly of cereal straws is greatly enhanced when feruloyl esterase activity is present, although the substrate specificity of the esterase appears to have some bearing on its optimal application. The involvement of feruloyl esterases in the improved enzymatic and microbial saccharification of cereal-derived material demonstrates a high importance for these enzymes in animal feed preparation and bioalcohol production.     

Evaluation of different microbial xylanolytic systems

The xylanolytic enzymes produced by Trichoderma reesei QM 9414, Aspergillus awamori VTT-D-75028, Fusarium oxysporum VTT-D-80134, Bacillus subtilis ATCC 12711 and Streptomyces olivochromogenes ATCC 21713 differed with respect to β-xylosidase activity and side-group cleaving activities. The highest xylanase activity was produced by T. reesei. All the fungi produced β-xylosidase, whereas in the bacterial culture filtrates β-xylosidase activity was negligible. T. reesei culture filtrate contained all the side-group cleaving activities assayed (acetyl esterase, α-glucuronidase and α-arabinosidase) and those of F. oxysporum and S. olivochromogenes contained esterase. All the side-group cleaving activities were low in the culture filtrates of A. awamori and B. subtilis.The differences between the xylanolytic systems were reflected in the hydrolysis of steamed birchwood hemicellulose. The xylose yields obtained ranged from 0 (with B. subtilis) to 90% (with T. reesei) of the theoretical maximum. The best enzyme for complete hemicellulose hydrolysis was therefore that of T. reesei. However, in some applications in which complete hydrolysis is not needed or in which hydrolysis of cellulose is to be avoided, one of the other xylanases may be more suitable than that of T. reesei.