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|Stability:||> 9 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-1,4-β-Xylanase|
|Assay Format:||Spectrophotometer (Semi-quantitative), Petri-dish (Qualitative)|
High purity dyed and crosslinked insoluble AZCL-Arabinoxylan (Wheat) for identification of enzyme activities in research, microbiological enzyme assays and in vitro diagnostic analysis.
Dyed and crosslinked wheat arabinoxylan. Substrate for the assay of endo-1,4-β-D-xylanase.
More insoluble chromogenic substrates available.
(Trichoderma longibrachiatum) E-XYAN4 - endo-1,4-β-Xylanase M4 (Aspergillus niger) E-XYRU6 - endo-1,4-β-Xylanase (rumen microorganism) E-XYNBS - endo-1,4-β-Xylanase
(Bacillus stearothermophilus T6) E-XYNACJ - endo-1,4-β-Xylanase (Cellvibrio japonicus) E-XYLNP - endo-1,4-β-Xylanase (Neocallimastix patriciarum) E-XYLATM - endo-1,4-β-Xylanase (Thermotoga maritima)
Evaluating Feruloyl Esterase-Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs.
Mkabayi, L., Malgas, S., Wilhelmi, B. S. & Pletschke, B. I. (2020). Agronomy, 10(5), 688.
Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids. The enzyme combination of 66% Xyn11 and 33% FAE5 or FAE6 (protein loading) produced the highest amounts of XOS, ferulic acid, and p-coumaric acid from untreated, hydrothermal, and acid pre-treated corn cobs. The combination of 66% Xyn11 and 33% FAE6 displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to Xyn11 alone), respectively. The hydrolysis product profiles revealed that xylobiose was the dominant XOS produced from untreated and pre-treated corn cobs. These results demonstrated that the efficient production of hydroxycinnamic acids and XOS from agricultural residues for industrial applications can be achieved through the synergistic action of FAE5 or FAE6 and Xyn11.Hide Abstract
Efficient expression and secretion of endo-1, 4-β-xylanase from Penicillium citrinum in non-conventional yeast Yarrowia lipolytica directed by the native and the preproLIP2 signal peptides.
Ouephanit, C., Boonvitthya, N., Theerachat, M., Bozonnet, S. & Chulalaksananukul, W. (2019). Protein Expression and Purification, 160, 1-6.
Filamentous fungi are the most common industrial xylanase producers. In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation affected the molecular mass of the recombinant xylanase, resulting in an apparent molecular weight larger than 60 kDa, whereas after deglycosylation, the recombinant XynA displayed a molecular mass of 20 kDa. The deglycosylated xylanase was purified by ion exchange chromatography and reached 185-fold of purification. The enzyme was optimally active at 55°C and pH 5 and stable over a broad pH range (3-9). It retained more than 80% of the original activity after 24 h. It conserved around 80% of the original activity after pre-incubation at 40°C for 6 h. With birchwood xylan as substrate, the enzyme showed a Km of 5.2 mg/mL, and kcat of 245 per s. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the re-XynA could be useful for variety of biotechnological applications.Hide Abstract
Novel xylanolytic triple domain enzyme targeted at feruloylated arabinoxylan degradation.
Holck, J., Djajadi, D. T., Brask, J., Pilgaard, B., Krogh, K. B., Meyer, A. S., Lange, L. & Wilkens, C. (2019). Enzyme and Microbial Technology, 129, 109353.
A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 α-l-arabinofuranosidase and a GH10 β-d-1,4-xylanase was identified in a metagenome obtained from wastewater treatment sludge. The capability of the CE1-GH62-GH10 multi-enzyme to degrade arabinoxylan was investigated to examine the hypothesis that CE1-GH62-GH10 would degrade arabinoxylan more efficiently than the corresponding equimolar mix of the individual enzymes. CE1-GH62-GH10 efficiently catalyzed the production of xylopyranose, xylobiose, xylotriose, arabinofuranose and ferulic acid (FA) when incubated with insoluble wheat arabinoxylan (WAX-I) (kcat = 20.8 ± 2.6 s−1). Surprisingly, in an equimolar mix of the individual enzymes a similar kcat towards WAX-I was observed (kcat = 17.3 ± 3.8 s−1). Similarly, when assayed on complex plant biomass the activity was comparable between CE1-GH62-GH10 and an equimolar mix of the individual enzymes. This suggests that from a hydrolytic point of view a CE1-GH62-GH10 multi-enzyme is not an advantage. Determination of the melting temperatures for CE1-GH62-GH10 (71.0 ± 0.05 °C) and CE1 (69.9 ± 0.02), GH62 (65.7 ± 0.06) and GH10 (71 ± 0.05 °C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost ˜50% activity within 2 h, while GH62 retained ˜50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained ˜50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (kcat/Km = 0.3 s−1 mg−1 ml) than the individual GH10 (kcat/Km = 0.12 s−1 ± 0.02 mg−1 ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower kcat towards WAX-I than GH62, which might be due to the presence of a putative carbohydrate binding module appended to CE1 at the N-terminal. Both CE1 and CE1-GH62 released insignificant amounts of FA from WAX-I, but FA was released from WAX-I when both CE1 and GH10 were present, which might be due to GH10 releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-α-l-Araf (kcat = 36.35 s−1). This suggests that CE1 preferably acts on soluble oligosaccharides.Hide Abstract
Identification of enzymes from genus Trichoderma that can accelerate formation of ferulic acid and ethyl ferulate in collaboration with rice koji enzyme in sake mash.
Ito, T., Sato, A., Takahashi, I., Ito, T., Takano, K., Noge, K., Okuda, M. & Hashizume, K. (2019). Journal of Bioscience and Bioengineering, 128(2), 177-182.
The enzymes responsible for acceleration of ferulic acid and ethyl ferulate formation in sake mash were studied. Ferulic acid and ethyl ferulate are formed during the sake brewing process from feruloylated glucuronoarabinoxylan. Cellulase reagent from genus Trichoderma was used instead of rice koji, because rice koji for sake brewing produces extremely low levels of xylan-degrading enzymes. A combination of the reagent with rice koji enzymes accelerated the formation of ferulic acid from α-rice powder. Addition of the reagent to sake mash increased ferulic acid and ethyl ferulate formation. The enzyme responsible for the accelerated formation was purified using a newly developed assay method and α-rice powder as a substrate. During the assay procedure, feruloylated oligosaccharide was converted to ferulic acid by feruloylesterase for HPLC analysis. Analysis of the N-terminal amino acid sequence of the purified samples was successfully conducted after pyroglutamyl aminopeptidase de-blocking. Purified enzymes were identified as members of the glycoside hydrolase family 10 (GH10) and family 11 (GH11) xylanases by BLASTP database research. The GH10 xylanase showed higher specific activity for α-rice powder and insoluble wheat arabinoxylan compared with GH11 xylanase; the GH11 xylanase showed higher specific activity for the other xylan substrates, especially glucuronoarabinoxylan. The GH10 xylanase showed higher accelerating activity than the GH11 xylanase in the sake mash. The results of this study provides useful knowledge on ferulic acid and ethyl ferulate formation in sake mash, the relative levels of these compounds and their influence on the sensory quality of sake.Hide Abstract
Steiner, J., Franke, K., Kießling, M., Fischer, S., Töpfl, S., Heinz, V. & Becker, T. (2018). Carbohydrate Polymers, 184, 315-322.
Brewer’s spent grain (BSG) constitutes various valuable carbohydrates that may contribute to a healthy diet. These components may be obtained from BSG via hydrothermal treatment (HT), a procedure for dissolving water-inextricable carbohydrates. The objective of this study was to investigate HT as an environmentally friendly technology for extracting high-molecular-weight fiber with proven beneficial effects on human health. Cellulose, β-glucan, and arabinoxylan (AX) served as model substances and were subjected to auto-hydrolysis at different temperatures and reaction times. The results were evaluated in terms of structural and chemical characteristics. When the treatment temperature was increased, the original weight-average molar mass of AX (370 kDa) and β-glucan (248 kDa) decreased gradually (<10 kDa), and the molar mass distribution narrowed. Further investigations focused on the heat-induced formation and elimination of monosaccharides and undesirable by-products. The concentrations of by-products were successfully described by kinetic models that can be used to optimize the hydrolysis process.Hide Abstract
Comino, P., Williams, B. A. & Gidley, M. J. (2018). Food & Function, In Press.
Insoluble dietary fibre is often considered to be fermented slower and to a lesser extent in (models for) the colon than soluble dietary fibre. However these comparisons are typically made for fibre components of different composition. In the case of fibre from refined cereal flours, there is little difference in fibre composition between soluble and insoluble forms, so effects of solubility on fermentation can be tested without this confounding factor. For each of wheat, rye, and hull-less barley, soluble and insoluble fibre fractions from refined flour and models for baking and extrusion had comparable in vitro fermentation rates and extents, with similar levels of short chain fatty acid metabolites. This study suggests that there should be little difference in the large intestinal nutritional functionality of the soluble and insoluble fibre fractions from cereal grain flours, either unprocessed or after baking or extrusion processing.Hide Abstract
Dilokpimol, A., Mäkelä, M. R., Mansouri, S., Belova, O., Waterstraat, M., Bunzel, M., de Vries, R. P. & Hildén, K. S. (2017). New Biotechnology, 37, 200-209.
A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10 mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3 mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass.Hide Abstract
Pitt, J. I., Lange, L., Lacey, A. E., Vuong, D., Midgley, D. J., Greenfield, P., Bradbury, M. I., Lacey, E., Busk, P. K., Pilgaard, B., Chooi, Y. H. & Piggott, A. M. (2017). PloS One, 12(4), e0170254.
Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.Hide Abstract
Wilkens, C., Busk, P. K., Pilgaard, B., Zhang, W. J., Nielsen, K. L., Nielsen, P. H. & Lange, L. (2017). Biotechnology for Biofuels, 10(1), 158
Background: Improved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry. Results: Enzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs. Conclusions: Our findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.Hide Abstract
Wefers, D., Cavalcante, J. J., Schendel, R. R., Deveryshetty, J., Wang, K., Wawrzak, Z., Mackie, R. I., Korotkin, N. M. & Cann, I. (2017). Journal of Molecular Biology, 429(16), 2509-2527.
Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12 mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98 Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.Hide Abstract
Huang, Y., Yi, Z., Jin, Y., Huang, M., He, K., Liu, D., Luo, H., Zhao, D., He, H., Fang, Y. & Zhao, H. (2017). Frontiers in Microbiology, 8, 1747.
Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.Hide Abstract
Guo, Z. P., Duquesne, S., Bozonnet, S., Nicaud, J. M., Marty, A. & O’Donohue, M. J. (2017). Biotechnology for Biofuels, 10(1), 298.
Background: A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate. Results: The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively. Conclusions: This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.Hide Abstract
Giacobbe, S., Vincent, F. & Faraco, V. (2014). New Biotechnology, 31(3), 230-236.
In this study, the α-L-arabinofuranosidase from Pleurotus ostreatus was subjected to directed evolution by expressing a library of around 7000 randomly mutated variants by error prone Polymerase Chain Reaction. High-throughput screening of the library for the most active variants was performed by assaying activity towards p-nitrophenyl α-L-arabinofuranoside, and a variant with higher activity than the wild type was selected, purified and characterised. It exhibited a Kcat of 7.3 × 103 ± 0.3 min-1, around 3-fold higher than that of the wild type (2.2 × 103 ± 0.2 min-1), and a KM (0.54 ± 0.06 mM) 30% lower than that of the wild type (0.70 ± 0.05 mM) towards this substrate. The mutant also showed improved catalytic properties towards pNP-β-D-glucopyranoside (Kcat of 50.85 ± 0.21 min−1 versus 11.0 ± 0.6 min-1) and it was shown able to hydrolyse larch arabinogalactan which is not recognised by the wild type. The mutant was also more active than the wild type towards arabinoxylan and was able to hydrolyse arabinan, which was not transformed by the wild type. The ability of rPoAbf F435Y/Y446F to hydrolyse these insoluble substrates expands its potential for application also to hemicelluloses, which in some types of pretreatment are recovered in solid fractions.Hide Abstract
Passerini, D., Coddeville, M., Le Bourgeois, P., Loubière, P., Ritzenthaler, P., Fontagné-Faucher, C., Daveran-Mingot, M. L. & Cocaign-Bousquet, M. (2013). Applied and Environmental Microbiology, 79(19), 5844-5852.
Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci.Hide Abstract
Baumann, M. J., Murphy, L., Lei, N., Krogh, K. B. R. M., Borch, K. & Westh, P. (2011). Analytical Biochemistry, 410(1), 19-26.
In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5 kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.Hide Abstract
Sørensen, A., Teller, P. J., Lübeck, P. S. & Ahring, B. K. (2011). Applied Biochemistry and Biotechnology, 164(7), 1058-1070.
Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.Hide Abstract
Eida, M. F., Nagaoka, T., Wasaki, J. & Kouno, K. (2011). Microbes Environ, 26(3), 220-227.
This study focused on the evaluation of cellulolytic and hemicellulolytic fungi isolated from sawdust compost (SDC) and coffee residue compost (CRC). To identify fungal isolates, the ITS region of fungal rRNA was amplified and sequenced. To evaluate enzyme production, isolates were inoculated onto wheat bran agar plates, and enzymes were extracted and tested for cellulase, xylanase, β-glucanase, mannanase, and protease activities using different azurine cross-linked (AZCL) substrates. In total, 18 isolates from SDC and 29 isolates from CRC were identified and evaluated. Four genera (Aspergillus, Galactomyces, Mucor, and Penicillium) and five genera (Aspergillus, Coniochaeta, Fusarium, Penicillium, and Trichoderma/Hypocrea) were dominant in SDC and CRC, respectively. Penicillium sp., Trichoderma sp., and Aspergillus sp. displayed high cellulolytic and hemicellulolytic activities, while Mucor isolates exhibited the highest β-glucanase and mannanase activities. The enzyme analyses revealed that Penicillium, Aspergillus, and Mucor isolates significantly contributed to the degradation of SDC, whereas Penicillium, Aspergillus, and Trichoderma isolates had a dominant role in the degradation of CRC. Notably, isolates SDCF5 (P. crustosum), CRCF6 (P. verruculosum), and CRCF2 and CRCF16 (T. harzianum/H. lixii) displayed high activity regarding cellulose and hemicellulose degradation, which indicates that these species could be beneficial for the improvement of biodegradation processes involving lignocellulosic materials.Hide Abstract
Phalip, V., Goubet, F., Carapito, R. & Jeltsch, J. M. (2009). Journal of Microbiology Biotechnology, 19(6), 573-581.
The complex enzyme pool secreted by the phytopathogenic fungus Fusarium graminearum in response to glucose or hop cell wall material as sole carbon sources was analyzed. The biochemical characterization of the enzymes present in the supernatant of fungal cultures in the glucose medium revealed only 5 different glycosyl hydrolase activities; by contrast, when analyzing cultures in the cell wall medium, 17 different activities were detected. This dramatic increase reflects the adaptation of the fungus by the synthesis of enzymes targeting all layers of the cell wall. When the enzymes secreted in the presence of plant cell wall were used to hydrolyze pretreated crude plant material, high levels of monosaccharides were measured with yields approaching 50% of total sugars released by an acid hydrolysis process. This report is the first biochemical characterization of numerous cellulases, hemicellulases, and pectinases secreted by F. graminearum and demonstrates the usefulness of the described protein cocktail for efficient enzymatic degradation of plant cell wall.Hide Abstract
Dornez, E., Gebruers, K., Wiame, S., Delcour, J. A. & Courtin, C. M. (2006). Journal of Agricultural and Food Chemistry, 54(22), 8521-8529.
To gain insight into the distribution of arabinoxylans (AX), endoxylanases, and endoxylanase inhibitors in industrial wheat roller milling, all streams, that is, 54 flour fractions, 4 bran fractions, and the germ, were analyzed for ash, starch, and protein contents, α-amylase activity levels, total (TOT-AX) and water-extractable arabinoxylan (WE-AX) contents, endoxylanase activity levels, and endoxylanase inhibitor (TAXI and XIP) contents. In general, bran fractions were significantly richer in TOT-AX and WE-AX contents, endoxylanase activity levels, and endoxylanase inhibitor contents than germ and, even more so, than flour fractions. In the 54 different flour fractions, minimal and maximal values for TOT-AX and WE-AX contents differed by ca. 2-fold, whereas they differed by ca. 15-fold for endoxylanase activity levels. The latter were positively correlated with ash and negatively correlated with starch content, suggesting that the endoxylanase activity in flour is strongly influenced by the level of bran contamination. TAXI contents in the flour fractions varied ca. 4-fold and were strongly correlated with bran-related parameters such as ash content and enzyme activity levels, whereas XIP contents varied ca. 3-fold and were not correlated with any of the parameters measured in this study. The results can be valuable in blending and optimizing wheat flour fractions to obtain flours with specific technological and nutritional benefits.Hide Abstract
Wejse, P. L., Ingvorsen, K. & Mortensen, K. K. (2003). Extremophiles, 7(5), 423-431.
The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca+2, Mn+2, Mg+2, Ba+2, Li+2, NaN3, and isopropanol was observed. The K m and V max values determined for Xyl 1 with 4-O-methyl-D-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.Hide Abstract