Purification and characterization of a XIP-type endoxylanase inhibitor from rice (Oryza sativa)

A rice XIP-type inhibitor was purified by affinity chromatography with an immobilized Aspergillus aculeatus family 10 endoxylanase. Rice XIP is a monomeric protein, with a molecular mass of ca. 32 kDa and a pI of ca. 5.6. Its N-terminal amino acid sequence was identical to that of a rice chitinase homologue, demonstrating the difficulty when using sequence information to differentiate between endoxylanase inhibitors and (putative) chitinases in rice. Rice XIP inhibited different endoxylanases to a varying degree. In particular, it most strongly inhibited family 10 endoxylanases from A. niger and A. oryzae, while several family 11 enzymes from Bacillus subtilis, A. niger and Trichoderma sp. were not sensitive to inhibition. The above mentioned A. aculeatus endoxylanase was not inhibited either, although gel permeation chromatography revealed that it complexed rice XIP in a 1:1 molar stoichiometric ratio.     

Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat

Two genes encoding family 11 endo-beta-1,4-xylanases (XylA, XylB) from Fusarium graminearum were cloned and expressed in Escherichia coli. The amount of active endoxylanase in the cytoplasmic soluble fraction was considerably improved by varying different expression parameters, including host strain and temperature during induction. Both recombinant endoxylanases showed a temperature optimum around 35 degrees C and neutral pH optima (around pH 7 and 8 for XylB and XylA, respectively). For the first time this allowed one to test endoxylanases of a phytopathogenic organism for inhibition by proteinaceous endoxylanase inhibitors TAXI and XIP. Whereas XylA and XylB were inhibited by TAXI-I, no inhibition activity could be detected upon incubation with XIP-I. The insensitivity of both F. graminearum endoxylanases towards XIP is surprising, since the latter is typically active against endoxylanases produced by (aerobic) fungi. As F. graminearum is an important phytopathogen, these findings have implications for the role of endoxylanase inhibitors in plant defence.     

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

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

Mutational analysis of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum reveals comprehensive insights into their inhibitor insensitivity

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

Molecular identification and chromosomal localization of genes encoding<Emphasis Type="Italic"> Triticum aestivum</Emphasis> xylanase inhibitor I-like proteins in cereals

TAXI (Triticum aestivum xylanase inhibitor) proteins are present in wheat flour and are known to inhibit glycosyl hydrolase family 11 endoxylanases, enzymes which are commonly applied in grain processing. Here, we describe the PCR-based molecular identification of genes encoding endoxylanase inhibitors HVXI and SCXI, the TAXI-like proteins from barley (Hordeum vulgare) and rye (Secale cereale) respectively. The HVXI coding sequence encodes a mature protein of 384 amino acids preceded by a 19 amino acid long signal sequence. SCXI-II/III has an open reading frame encoding a signal peptide of 21 amino acids and a mature protein of 375 amino acids. As for TAXI-I, no introns were detected in the untranslated regions and coding sequences identified. These newly identified sequences allowed us to perform a multiple sequence alignment with TAXI-I and similar proteins. Rice TAXI-type proteins clustered together with the cereal endoxylanase inhibitors. Dicotyledonous proteins with sequence similarity to TAXI-I, including the tomato xyloglucan-specific endoglucanase inhibiting protein, formed a different clade. The TAXI-type proteins may hence be part of a superfamily of proteins all involved in plant responses to biotic or abiotic stress and for which a function as glycosyl hydrolase inhibitors can be suggested. The chromosomal localization of the TAXI-I gene identified on wheat chromosome 3B, of the SCXI-II/III gene identified on rye chromosome 6R, and the presence of a cluster of TAXI-like genes on rice chromosome 1, allowed us to assign the location of TAXI-like genes to the wheat-rye translocation area 3BL/6RL characterized by RFLP markers XGlb33 and Xpsr454 and isozyme Est-5. In rice, RFLP marker C1310S corresponds to a TAXI-like protein encoding sequence.Communicated by P. Langridge     

Molecular and biochemical characterization of a novel intracellular low-temperature-active xylanase

A 990 bp full-length gene (xynAHJ2) encoding a 329- residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperatureactive enzyme (exhibiting optimum activity at 35 degrees C, 62% at 20 degrees C, and 38% at 10 degrees C; thermolability at > or =45 degrees C). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to Ni2+, Ca2+, or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperatureactive xylanase.     

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides

An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents. It was identified as Bacillus halodurans. The purified xylanase was active from pH 7 to 12 and 30 to 100°C with optimal activity at pH 9.0 and 80°C. It had T_1/2 values of 40 and 15 min at 70 and 80°C, respectively. Activity was stimulated by dithiothreitol but strongly inhibited by N-bromosuccinimide. Its action on birchwood xylan and agro-residues liberated xylooligosaccharides of 2–7 degree of polymerization, and thus, the mode of action is similar to endoxylanases of the family 10 glucoside hydrolases.     

Purification of TAXI-like Endoxylanase Inhibitors from Wheat (Triticum Aestivum L.) Whole Meal Reveals a Family of Iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues

An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and β-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0–10.0) and temperatures (30–100 °C) with optima at pH 7.5 and 70 °C, while β-xylosidase is optimally active at pH 7.0 and 60 °C. The T _1/2 values of the endoxylanase and β-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward’s reagent K. N-BS and DEPC strongly inhibited β-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of β-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and β-xylosidase liberated xylose as main hydrolysis product.     

Identification and characterization of a novel arabinoxylanase from wheat flour.

An endogenous wheat (Triticum aestivum) flour endoxylanase was purified to homogeneity from a crude wheat flour extract by ammonium sulfate precipitation and cation-exchange chromatography. The 30-kD protein had an isoelectric point of 9.3 or higher. A sequence of 19 amino acids at the NH2 terminus showed 84.2% identity with an internal sequence of 15-kD grain-softness protein, friabilin. High-performance anion-exchange chromatography and gel-permeation analysis of the hydrolysis products indicated the preferential hydrolysis of highly branched structures by the enzyme; wheat arabinoxylan and rye (Secale cereale) arabinoxylan (high arabinose to xylose ratios) were hydrolyzed more efficiently by this enzyme than oat (Avena sativa) spelt xylan (low arabinose to xylose ratios). The release of the hydrolysis products as a function of time suggested that the endoxylanolytic activity was associated with the release of arabinose units from the polysaccharides, suggesting that the enzyme action is similar to that by endoxylanases from Ceratocystis paradoxa, Aspergillus niger, and Neurospora crassa. Although the enzyme released arabinose from arabinoxylan, it did not hydrolyze p-nitrophenyl-alpha-L-arabinofuranoside. From the above, it follows that the enzyme, called arabinoxylanase, differs from most microbial endoxylanases and from an endoxylanase purified earlier from wheat flour.     

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

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

Purification and characterization of endoxylanase Xln-1 from <Emphasis Type="Italic">Aspergillus niger</Emphasis> B03

An extracellular endoxylanase was isolated from the xylanolytic complex of Aspergillus niger B03. The enzyme was purified to a homogenous form using consecutive ultrafiltration and anion exchange chromatography. The endoxylanase was a monomer protein with a molecular weight of 33,000 Da determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 34,000 Da determined by gel filtration. The optimal pH and temperature values for the enzyme action were 6.0 and 60°C, respectively. Endoxylanase was stable at 40°C, pH 7.0 for 210 min. The thermal stability of the enzyme was significantly increased in the presence of glycerol and sorbitol. The enzyme activity was inhibited by Cu²⁺, Fe²⁺, Fe³⁺, and Ag¹⁺, and it was activated by Mn²⁺. The substrate specificity and kinetic parameters of the enzyme were determined with different types of xylans. Endoxylanase displayed maximum activity in the case of oat spelt xylan, with an apparent K _m value of 8.19 mg/ml. The substrate specificity and the product profile of the enzyme suggested it to be an endoxylanase.     

Functional display of family 11 endoxylanases on the surface of phage M13

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.     

Two extracellular endoxylanases from alkaliphilic Bacillus firmus differ in their synthesis

To investigate the synthesis of two extracellular endoxylanases, xylan-binding and unbound xylanases from an alkaliphilic Bacillus firmus, washed cells were incubated in alkaline mineral salt media containing various carbon sources. The 23 kDa xylan-binding endoxylanase (XBE), which hydrolyses insoluble xylan, was produced before the 45 kDa, unbound endoxylanase. All the carbon sources tested at 5 mg ml^–1, including glucose, induced production of XBE but the unbound xylanase was totally repressed by glucose. The production of XBE increased when glucose concentration increased but was not synthesized until the glucose in the medium was less than 1 mg ml^–1.     

Proteinaceous inhibitors of microbial xylanases

At the end of 1990s two structurally different proteinaceous inhibitors of xylanases were discovered in the grain of wheat (Triticum aestivum). They were named TAXI (T. aestivum xylanase inhibitor) and XIP (xylanase-inhibiting protein). Later it was shown that TAXI and XIP in wheat are present in several isoforms encoded by different genes. TAXI- and XIP-like inhibitors have also been found in other cereals-barley, rye, rice, maize, etc. All these proteins can specifically inhibit activity of fungal and bacterial xylanases belonging to families 10 and 11 of glycoside hydrolases, but they do not affect endogenous enzymes produced by plants. A common viewpoint is that the presence of proteinaceous inhibitors in cereals is a response of plants to pathogenic attack by microorganisms. A few years ago, an inhibitor of a third type was discovered in wheat. It was named TLXI (thaumatin-like xylanase inhibitor) because of its similarity to the thaumatin family of plant proteins. In this review, the occurrence of proteinaceous inhibitors of xylanases in different cereals, their specificity towards fungal and bacterial enzymes, as well as structural features responsible for enzyme sensitivity to various types of inhibitors are discussed.     

Production of the <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> endo-1,4-β-mannanase in <Emphasis Type="Italic">A. niger</Emphasis>

The β-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of β-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd _P) and the A. awamori glucoamylase terminator (glaA_T). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml⁻¹ and 574 nkat mg⁻¹ dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.     

Characterization of a xylanase inhibitor TAXI-I from wheat

Xylanase inhibitor TAXI-I gene was cloned from wheat (Triticum aestivum L.) and then TAXI-I encoding sequence was expressed in Escherichia coli. The recombinant TAXI-I protein inhibited glycoside hydrolase (GH) family 11 xylanases in Aspergillus niger (Anx; a fungal xylanase), and Thermomonospora fusca (Tfx; a bacterial xylanase), and also inhibited hybrid xylanases Atx (a hybrid xylanase whose parents are T. fusca and A. niger) and Btx (a hybrid xylanase whose parents are T. fusca and Bacillus subtilis). Among the tested xylanases, A. niger xylanase was the most inhibited one by wheat xylanase inhibitor TAXI-I, while T. fusca xylanase was the least inhibited one. The profile of TAXI-I gene expression in wheat in response to phytohormones was also investigated. TAXI-I gene expression was drastically induced by methyl jasmonate (MeJa), and hardly detected in gibberellic acid (GA) treatment. Therefore, TAXI-I might be involved in plant defense against fungal and bacteria xylanases.