A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase
McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.
The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
Novel surface-based methodologies for investigating GH11 xylanase–lignin derivative interactions.
Zeder-Lutz, G., Renau-Ferrer, S., Aguié-Béghin, V., Rakotoarivonina, H., Chabbert, B., Altschuh, D. & Rémond, C. (2013). Analyst, 138(22), 6889-6899.
The recalcitrance of lignocellulose to bioprocessing represents the core problem and remains the limiting factor in creating an economy based on lignocellulosic ethanol production. Lignin is responsible for unproductive interactions with enzymes, and understanding how lignin impairs the susceptibility of biomass to enzymatic hydrolysis represents a significant aim in optimising the biological deconstruction of lignocellulose. The objective of this study was to develop methodologies based on surface plasmon resonance (SPR), which provide novel insights into the interactions between xylanase (Tx-xyn11) and phenolic compounds or lignin oligomers. In a first approach, Tx-xyn11 was fixed onto sensor surfaces, and phenolic molecules were applied in the liquid phase. The results demonstrated weak affinity and over-stoichiometric binding, as several phenolic molecules bound to each xylanase molecule. This approach, requiring the use of soluble molecules in the liquid phase, is not applicable to insoluble lignin oligomers, such as the dehydrogenation polymer (DHP). An alternative approach was developed in which a lignin oligomer was fixed onto a sensor surface. Due to their hydrophobic properties, the preparation of stable lignin layers on the sensor surfaces represented a considerable challenge. Among the various chemical and physico-chemical approaches assayed, two approaches (physisorption via the Langmuir–Blodgett technique onto self-assembled monolayer (SAM)-modified gold and covalent coupling to a carboxylated dextran matrix) led to stable lignin layers, which allowed the study of its interactions with Tx-xyn11 in the liquid phase. Our results indicated the presence of weak and non-specific interactions between Tx-xyn11 and DHP.
Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi.
Vršanská, M., Kolenová, K., Puchart, V. & Biely, P. (2007). FEBS Journal, 274(7), 1666-1677.
The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI-TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear β-1,4-xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA3Xyl3 (aldotetraouronic acid), MeGlcA3Xyl4 (aldopentaouronic acid) and MeGlcA3Xyl5 (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA2Xyl2, MeGlcA2Xyl3 and MeGlcA2Xyl4. As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3–14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with β-xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA2Xyl2. In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.
Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of pseudomonas cellulosa xylanase 10A.
Andrews, S. R., Charnock, S. J., Lakey, J. H., Davies, G. J., Claeyssens, M., Nerinckx, W., Underwood, M., Sinnott, M. L., Warren, R. A. J. & Gilbert, H. J. (2000). Journal of Biological Chemistry, 275(30), 23027-23033.
The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes β1,4-linked xylans but exhibits very low activity against aryl-β-cellobiosides. The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-β-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the –1 and –2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751–4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the –2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the –1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-β-cellobioside and 4-nitrophenyl-β-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-β-xylobioside activity but did not significantly reduce 4-nitrophenyl-β-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.
In vitro fermentation of cereal dietary fibre carbohydrates by probiotic and intestinal bacteria.
Crittenden, R., Karppinen, S., Ojanen, S., Tenkanen, M., Fagerström, R., Mättö, J., Saarela, M., Mattila-Sandholm, T. & Poutanen, K. (2002). Journal of the Science of Food and Agriculture, 82(8), 781-789.
A range of probiotic and other intestinal bacteria were examined for their ability to ferment the dietary fibre carbohydrates β-glucan, xylan, xylo-oligosaccharides (XOS) and arabinoxylan. β-Glucan was fermented by Bacteroides spp and Clostridium beijerinckii but was not fermented by lactobacilli, bifidobacteria, enterococci or Escherichia coli. Unsubstituted xylan was not fermented by any of the probiotic bacteria examined. However, many Bifidobacterium species and Lactobacillus brevis were able to grow to high yields using XOS. XOS were also efficiently fermented by some Bacteroides isolates but not by E coli, enterococci, Clostridium difficile, Clostridium perfringens or by the majority of intestinal Lactobacillus species examined. Bifidobacterium longum strains were able to grow well using arabinoxylan as the sole carbon source. These organisms hydrolysed and fermented the arabinosyl residues from arabinoxylan but did not substantially utilise the xylan backbone of the polysaccharide. Arabinoxylan was not fermented by lactobacilli, enterococci, E coli, C perfringens or C difficile and has potential to be an applicable carbohydrate to complement probiotic Bif longum strains in synbiotic combinations.
Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0.
Bataillon, M., Nunes Cardinali, A. P., Castillon, N. & Duchiron, F. (2000). Enzyme and Microbial Technology, 26(2), 187-192.
A Bacillus spp. strain SPS-0, isolated from a hot spring in Portugal, produced an extracellular xylanase upon growth on wheat bran arabinoxylan. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange, gel filtration, and affinity chromatography. The optimum temperature and pH for activity was 75°C and 6.0. Xylanase was stable up to 70°C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg+2 ions. β-Mercaptoethanol, dithiothreitol, and Mn+2 stimulated the xylanase activity. The products of birchwood xylan hydrolysis were xylose, xylobiose, xylotriose, and xylotetraose. Kinetic experiments at 60°C and pH 6.0 gave Vmax and KM values of 2420 nkat/mg and 0.7 mg/ml.
Novel bifunctional α-L-arabinofuranosidase/xylobiohydrolase (ABF3) from Penicillium purpurogenum.
Ravanal, M. C., Callegari, E. & Eyzaguirre, J. (2010). Applied and Environmental Microbiology, 76(15), 5247-5253.
The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl α-L-arabinofuranoside and p-nitrophenyl β-D-xylopyranoside with a Km of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both α-L-arabinofuranosidase and xylobiohydrolase functionalities.
Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module.
Gunnarsson, L. C., Montanier, C., Tunnicliffe, R. B., Williamson, M. P., Gilbert, H. J., Nordberg, K. E. & Ohlin, M. (2007). Biochem. J, 406(2), 209-214.
Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.
The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains.
Paradis, F. W., Zhu, H., Krell, P. J., Phillips, J. P. & Forsberg, C. W. (1993). Journal of Bacteriology, 175(23), 7666-7672.
The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen.
Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans.
Moreau, A., Roberge, M., Manin, C., Shareck, F., Kluepfel, D. & Morosoli, R. (1994). Biochem. J, 302, 291-295.
On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl β-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl β-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.
Penicillium purpurogenum produces two GH family 43 enzymes with β-xylosidase activity, one monofunctional and the other bifunctional: Biochemical and structural analyses explain the difference.
Ravanal, M. C., Alegría-Arcos, M., Gonzalez-Nilo, F. D. & Eyzaguirre, J. (2013). Archives of Biochemistry and Biophysics, 540(1), 117-124.
β-Xylosidases participate in xylan biodegradation, liberating xylose from the non-reducing end of xylooligosaccharides. The fungus Penicillium purpurogenum secretes two enzymes with β-D-xylosidase activity belonging to family 43 of the glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-α-L-arabinofuranoside (pNPAra) and p-nitrophenyl-β-D-xylopyranoside (pNPXyl) with a KM of 0.65 and 12 mM, respectively. The other, β-D-xylosidase 1 (XYL1), is only active on pNPXyl with a KM of 0.55 mM. The xyl1 gene was expressed in Pichia pastoris, purified and characterized. The properties of both enzymes were compared in order to explain their difference in substrate specificity. Structural models for each protein were built using homology modeling tools. Molecular docking simulations were used to analyze the interactions defining the affinity of the proteins to both ligands. The structural analysis shows that active complexes (ABF3–pNPXyl, ABF3–pNPAra and XYL1–pNPXyl) possess specific interactions between substrates and catalytic residues, which are absent in the inactive complex (XYL1–pNPAra), while other interactions with non-catalytic residues are found in all complexes. pNPAra is a competitive inhibitor for XYL1 (Ki = 2.5 mM), confirming that pNPAra does bind to the active site but not to the catalytic residues.
Xylan-degrading enzymes from Aspergillus terreus: Physicochemical features and functional studies on hydrolysis of cellulose pulp.
de Souza Moreira, L. R., Álvares, A. D. C. M., da Silva Jr, F. G., de Freitas, S. M. & Ferreira Filho, E. X. (2015). Carbohydrate polymers, 134, 700-708.
Two endo-β-1,4-xylanases named XylT1 and XylT2, previously purified from Aspergillus terreus, were structurally investigated by fluorescence quenching and characterized with respect to their binding properties with phenolic compounds. Neutral and charged quenchers had access to both enzymes in neutral and alkaline pHs. The greatest access was noted for the negative quencher, possibly due to positive amino acid residues in the vicinity of tryptophan. These tryptophan environments may partially explain the conformational differences and lower binding constants of phenolic compounds for XylT2 than XylT1. These results show that xylanases present structural and functional differences, despite belonging to similar families. XylT1 and XylT2 were also evaluated for their ability to hydrolyze cellulose pulp in different stages of bleaching. Both enzymes promoted hydrolysis of cellulose pulps, which was confirmed by the release of total reducing sugars, pentoses and chromophoric material. Analysis of released xylooligosaccharides demonstrated a preferential release of xylobiose. None of xylanases released glucose, showing that they do not hydrolyze the cellulose present in the pulp, making both enzymes excellent choices for bio-bleaching applications.
Enzymatic hydrolysis of hemicelluloses from Miscanthus to monosaccharides or xylo-oligosaccharides by recombinant hemicellulases.
Li, H., Xue, Y., Wu, J., Wu, H., Qin, G., Li, C., Ding, J., Liu, J., Gan, L. & Long, M. (2016). Industrial Crops and Products, 79, 170-179.
Hemicelluloses isolated from holocellulose (MHOC) from Miscanthus were characterized by RP-HPLC-UV, FT-IR, and NMR. The hemicelluloses and recombinant hemicellulases, including endo-β-1,4-xylanases (HoXyn11A and AnXyn10C), β-xylosidases (AnXln3D), and α-L-arabinofuranosidases (AnAxh62A), as well as their interaction mechanisms were investigated by enzymatic hydrolysis. AnXyn10C released shorter end products than HoXyn11A from isolated hemicelluloses (IHEC). AnAxh62A was able to release all single-substituted α-L-arabinofuranosyl residues from IHEC. AnXyn10C and HoXyn11A were able to directly act on MHOC, whereas AnAxh62A and AnXln3D did not. The combination of HoXyn11A and AnAxh62A produced the highest xylo-oligosaccharides (XOS) yield from IHEC, whereas AnXyn10C alone produced the highest XOS yield from MHOC. The combination of HoXyn11A, AnAxh62A, and AnXln3D achieved the highest xylose yield from IHEC, whereas the combination of AnXyn10C, AnAxh62A, and AnXln3D achieved the highest xylose yield from MHOC. This study contributes to the development of efficient enzyme cocktails for the bioconversion of hemicelluloses from Miscanthus into monosaccharides and XOS.
Systematic evaluation of the degraded products evolved from the hydrothermal pretreatment of sweet sorghum stems.
Sun, S., Wen, J., Sun, S. & Sun, R. C. (2015). Biotechnology for biofuels, 8(1), 37.
Background: Conversion of plant cell walls to bioethanol and bio-based chemicals requires pretreatment as a necessary step to reduce recalcitrance of cell walls to enzymatic and microbial deconstruction. In this study, the sweet sorghum stems were subjected to various hydrothermal pretreatment processes (110°C to 230°C, 0.5 to 2.0 h), and the focus of this work is to systematically evaluate the degraded products of polysaccharides and lignins in the liquor phase obtained during the pretreatment process. Results: The maximum yield of xylooligosaccharides (52.25%) with a relatively low level of xylose and other degraded products was achieved at a relatively high pretreatment temperature (170°C) for a short reaction time (0.5 h). Higher temperature (>170°C) and/or longer reaction time (>0.5 h at 170°C) resulted in a decreasing yield of xylooligosaccharides, but increased the concentration of arabinose and galactose. The xylooligosaccharides obtained are composed of xylopyranosyl residues, together with lower amounts of 4-O-Me-α-D-GlcpA units. Meanwhile, the concentrations of the degraded products (especially furfural) increased as a function of pretreatment temperature and time. Molecular weights of the water-soluble polysaccharides and lignins indicated that the degradation of the polysaccharides and lignins occurred during the conditions of harsh hydrothermal pretreatment. In addition, the water-soluble polysaccharides (rich in xylan) and water-soluble lignins (rich in β-O-4 linkages) were obtained at 170°C for 1.0 h. Conclusions: The present study demonstrated that the hydrothermal pretreatment condition had a remarkable impact on the compositions and the chemical structures of the degraded products. An extensive understanding of the degraded products from polysaccharides and lignins during the hydrothermal pretreatment will be beneficial to value-added applications of multiple chemicals in the biorefinery for bioethanol industry.
Isolation and divalent-metal activation of a β-xylosidase, RUM630-BX.
Jordan, D. B., Braker, J. D., Wagschal, K., Stoller, J. R. & Lee, C. C. (2016). Enzyme and microbial technology, 82, 158-163.
The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg2+, Mn2+ and Co2+ but not by Ca2+, Ni2+, and Zn2+. Activation of RUM630-BX by Mg2+ (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s-1 mM-1 on xylotetraose being the highest reported.
Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation.
Marcolongo, L., La Cara, F., Morana, A., Di Salle, A., Del Monaco, G., Paixão, S. M., Alves, L. & Ionata, E. (2015). World Journal of Microbiology and Biotechnology, 31(4), 633-648.
An extracellular thermo-alkali-stable and cellulase-free xylanase from Geobacillus thermodenitrificans A333 was purified to homogeneity by ion exchange and size exclusion chromatography. Its molecular mass was 44 kDa as estimated in native and denaturing conditions by gel filtration and SDS-PAGE analysis, respectively. The xylanase (GtXyn) exhibited maximum activity at 70°C and pH 7.5. It was stable over broad ranges of temperature and pH retaining 88 % of activity at 60°C and up to 97 % in the pH range 7.5-10.0 after 24 h. Moreover, the enzyme was active up to 3.0 M sodium chloride concentration, exhibiting at that value 70 % residual activity after 1 h. The presence of other metal ions did not affect the activity with the sole exceptions of K+ that showed a stimulating effect, and Fe2+, Co2+ and Hg2+, which inhibited the enzyme. The xylanase was activated by non-ionic surfactants and was stable in organic solvents remaining fully active over 24 h of incubation in 40 % ethanol at 25°C. Furthermore, the enzyme was resistant to most of the neutral and alkaline proteases tested. The enzyme was active only on xylan, showing no marked preference towards xylans from different origins. The hydrolysis of beechwood xylan and agriculture-based biomass materials yielded xylooligosaccharides with a polymerization degree ranging from 2 to 6 units and xylobiose and xylotriose as main products. These properties indicate G. thermodenitrificans A333 xylanase as a promising candidate for several biotechnological applications, such as xylooligosaccharides preparation.
Structural insights into the inhibition of cellobiohydrolase Cel7A by xylo‐oligosaccharides.
Momeni, M. H., Ubhayasekera, W., Sandgren, M., Ståhlberg, J. & Hansson, H. (2015). The FEBS journal, 282(11), 2167-2177.
The filamentous fungus Hypocrea jecorina (anamorph of Trichoderma reesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides (XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family 7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H. jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted ~ 2.4 Å towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition.
An immobilized bifunctional xylanase on carbon-coated chitosan nanoparticles with a potential application in xylan-rich biomass bioconversion.
Liu, M. Q., Huo, W. K., Xu, X. & Jin, D. F. (2015). Journal of Molecular Catalysis B: Enzymatic, 120, 119-126.
Immobilization technology offers many enzymatic advantages and overcomes the limitations of free enzymes. Bi- or multifunctional enzymes for industrial use have elicited much interest in recent years. The present work reported that a novel carbon nanoparticle-based supports was prepared by layer-by-layer self-assemble approach. The constructed bifunctional enzyme (ATXX) was successfully immobilized on the supports by covalent bonds. The prepared carbon-coated chitosan nanoparticles showed high binding capacity of about 289.9 mg g-1-particles for ATXX. The Michaelis-Menten constants (Km) and maximal activity (Vmat) of immobilized ATXX were 4.83 mg ml-1 and 67.42 µmol min-1 mg-1-particles (xylanase activity), as well as 6.13 mg ml-1 and 17.92 µmol min-1 mg-1-particles (cellulase activity), respectively. The immobilized ATXX showed improved thermostability and storage stability compared with the free enzyme. The immobilized ATXX retained 82.6% xylanase activity after seven successive reactions. High-performance liquid chromatography (HPLC) analysis revealed that xylobiose (X2) was the main hydrolysis product released from beechwood xylan, birchwood xylan, and oat spelt xylan by immobilized ATXX. Wheat bran and wheat bran insoluble xylan could be directly hydrolyzed by immobilized ATXX, which demonstrated a potential use for xylan bioconversion to xylooligosaccharides by the immobilized ATXX.
Separation of xylose oligomers from autohydrolyzed Miscanthus × giganteus using centrifugal partition chromatography.
Chen, M. H., Rajan, K., Carrier, D. J. & Singh, V. (2015). Food and Bioproducts Processing, 95, 125-132.
Autohydrolysis of cellulosic materials for saccharification generates xylose-oligosaccharides (XOS), due to the partial hydrolysis of xylan. Developing an efficient method for the separation and recovery of XOS from the prehydrolyzates would provide an excellent opportunity for the better utilization of the cellulosic material and for value-added co-product production. In this study, we investigated the use of centrifugal partition chromatography (CPC) for the fractionation of XOS from Miscanthus × giganteus (M × G). During autohydrolysis of miscanthus biomass at 180°C for 20 min, 63% of xylan was converted into XOS and xylose. The ensuing XOS concentrate contained up to 30% of XOS, which were distributed as 15.9% xylobiose (DP2), 5.9% xylotriose, (DP3), 5.6% xylotetraose (DP4), 0.8% xylopentaose (DP5) and 0.6% xylohexaose (DP6). The XOS concentrate was further fractionated by CPC with a solvent system composed of 4:1:4 (v/v/v) butanol:methanol:water. Using CPC techniques, 230 mg (80%) of DP2 to DP6 oligomers were fractionated from 1 g of XOS concentrate. The recoveries of individual XOS were 90.2% DP2, 64.5% DP3, 71.2% DP4, 61.9% DP5 and 68.9% DP6. The purities of DP2 to DP6 fractions were 61.9%, 63.2%, 44.5%, 31.5% and 51.3%, respectively. Presence of DP2 and DP3 in the CPC purified fractions was further validated by mass spectrometry analysis. The study provided information on fast recovery of individual XOS from crude biomass prehydrolyzate.