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.     

Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display

Specific binding of interacting proteins generally depends on a limited set of amino acid residues located at the contact interface. We have applied a phage-display-based screening method to simultaneously evaluate the role of multiple residues of endo-beta-1,4-xylanase enzymes in conferring binding specificity towards two different endoxylanase inhibitors. Seven residues of the two beta-strand 'thumb' region of Trichoderma longibrachiatum endo-beta-1,4-xylanase XynII were targeted for randomization. The generated combinatorial library representing 62,208 site-directed variants was displayed on the surface of filamentous phage and selected against xylanase inhibitor protein (XIP) and Triticum aestivum xylanase inhibitor (TAXI). DNA sequence analysis of phagemid panning isolates provided information on the occurrence of particular amino acids at distinct positions. In particular, residues at positions 124 (Asn) and 131 (Thr) were found to be critical for specific inhibitor binding. These residue predictions derived from the combinatorial exploration of the thumb region and accompanying sequence analyses were experimentally confirmed by testing the inhibitor sensitivity of a limited set of recombinantly expressed XynII mutants. In addition, we successfully altered the inhibition susceptibility of the bacterial Bacillus subtilis endoxylanase XynA from XIP-insensitive to XIP-sensitive.     

Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Production of a heterologous endo-1,4-beta-xylanase in the yeast Pichia stipitis with an O(2)-regulated promoter

The Cryptococcus albidus XLN-gene (encoding endo-1,4-β-xylanase) was expressed in the yeast Pichia stipitis under the control of the PsADH2-promoter, which is activated under O₂ limitation. The resulting transformant produced endo-1,4-β-xylanase after a shift to anoxic conditions. Endo-1,4-β-xylanase production was enhanced by limited aeration after the shift.     

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.     

Sequence analysis,cloning and over-expression of an endoxylanase from the alkaliphilic <Emphasis Type="BoldItalic">Bacillus halodurans</Emphasis>

The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-β-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg⁻¹.     

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

To isolate phages displaying a practical and useful antibody with a high k_on value and/or a low k_off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen (k_on′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen (k_off′), the effective antigen concentration (C), the time for the binding process (t_b) and the time for the washing process (t_w). An optimum set of operating parameters (C, t_b and t_w) for isolating phages displaying an antibody with a high k_on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k_on value and one displayed an antibody with a 30-fold greater k_on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.     

Screening of a xylanase clone from a fosmid library of rumen microbiota in hu sheep

The glycosyl hydrolase family 11, which is responsible for carbohydrate metabolism, was identified in the open reading frame (ORF) 6 of a xylanase positive clone from a fosmid library of rumen microbiota of Hu sheep. A BLASTP search of GenBank revealed that ORF6 encoded a 355-amino acid putative endoxylanase, having 61% similarity (e(-73)) to endo-1,4-β-xylanase of Fibrobacter succinogenes S85 (YP_003250510.1). Predicted with the SWISS-MODEL, there were two separate β-sandwich clusters linked with a high serine containing linker in ORF6. The N-terminal β-sandwich is a novel endoxylanase of the glycosyl hydrolase family 11 with a specific activity of 1150.00?U/mg. The optimal pH and temperature for this enzyme were shown to be pH 5.0 and 50°C, respectively. The C-terminal helped increase the stability of the xylanase but decreased the activity to some degree. The C-terminal β-sandwich could bind avicel, but no conserved domain could be found. It may be a novel carbohydrate-binding module.     

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.     

宇佐美曲霉木聚糖酶基因xynⅡ在不同毕赤酵母中的分泌表达

将宇佐美曲霉E001的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K中,得到重组质粒pPXY-NII,将其经SalⅠ线性化后分别转化2株毕赤酵母GS115和KM71,xynⅡ基因通过同源重组被整合到毕赤酵母染色体上,并处于酵母α因子的下游,经筛选获得阳性重组菌PXGL98(Mut+)和PXKL29(Muts)。该木聚糖酶基因在2株毕赤酵母中均实现了分泌表达。同时对工程菌的发酵条件进行了优化,在甲醇诱导下,PXGL98与PXKL29培养物上清液中的酶活力分别可达1156.92 U/mL和1646.03 U/mL。     

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.     

Protein homology modeling,docking, and phylogenetic analyses of an endo-1,4-β-xylanase GH11 of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

Endo-1,4-β-xylanase (EC 3.2.1.8) is a crucial enzyme that randomly cleaves the β-1,4-glycosidic linkages of the xylan backbone, releasing xylooligomers of different lengths. The three-dimensional structure of the endo-β-1,4-xylanase protein (xyl1) from Colletotrichum lindemuthianum was modeled and docked with various xylan model compounds. Docking analyses revealed significantly higher stability of C. lindemuthianum XYL1 with the xylopentaose oligomer. Residues interacting with the model oligomers at the respective enzyme active sites were found to be in accord with their role in xylan degradation. Nevertheless, docking analyses of xylanases GH11 from Colletotrichum sp. revealed significative differences in structure, integration of the substrate into the active site, and in the glutamate residues of the catalytic site involved in substrate hydrolysis; of these proteins, 36%, 60%, and 4% integrated xylotetraose, xylopentaose, and xylohexaose in the active site, respectively. Since endoxylanases GH11 from Colletotrichum species interact much more efficiently with xylopentaose and xylotetraose, and xylanases GH11 from different fungi do not seem to have the same substrate binding subsites, we propose that they are enzymes with different affinity to xylooligosaccharides. In agreement with this idea, phylogenetic analyses of xylanases from Colletotrichum sp. show four lineages, suggesting diversifying selection. Most likely, the polydiversity or structural polymolecularity of xylan in plant cell walls processed by these organisms play a determinant role in diversifying selection.     

Russian Article pp. 191–195

Three cellulase components and one xylanase of Trichoderma sp. M-17 have been immobilzed on a soluble high molecular weight polymer (PVA), using carbodiimide. The immobilized enzymes retained about 80% of the cellulase, cellulose 1,4-β-cellobiosidase, β-glucosidase and 60% endo-1,4-β-xylanase activities. The bound enzymes catalyzed the hydrolysis of alkali-treated cornstalks with a higher efficiency than the free cellulase. The potential for reutilization of the immobilized enzymes was studied using membrane filters and the system was found to be active for three cycles.     

短小芽孢杆菌木聚糖酶基因在毕赤酶母中的分泌表达及酶学性质研究

将短小芽孢杆菌HB030的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K,得到重组质粒pH-BM220,将pHBM220经酶切后分别转化三株毕赤酵母KM71、GS115、SMD1168,该木聚糖酶基因在三株毕赤酵母中均实现了分泌表达,将重组毕赤酵母KM71（pHBM220）,GS115（pHBM220),GS115(pHBM220),SMD1168（pHBM220）分别诱导产酶,对重组酶进行相关的酶学性质分析表明,三的最适反应pH值约为5.5,最适反应温度约为60℃,在其最适反应条件下测得三粗酶液酶活分别为10.80IU/mL,11.63IU/mL,9.68IU/mL,重组毕赤酵母KM71（pHBM220）所产酶的热稳定性较好,而在pH稳定性方面三没有太大的差异。     

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.     

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.     

Optimization of the production and molecular characterization of cellulase-free xylanase from an alkalophillic Bacillus subtilis SD8 isolated from paper mill effluent

Applied Biochemistry and Microbiology - Culture conditions for the production of cellulase-free endo-β-1,4-xylanase by an alkalophilic strain of Bacillus subtilis SD8 (BsSD8-xylanase) isolated...     

Extracellular Carbohydrate Esterase from the Basidiomycete Coprinopsis cinerea Released Ferulic and Acetic Acids from Xylan

cDNA encoding an extracellular carbohydrate esterase (CcEst1) was cloned from the basidiomycete Coprinopsis cinerea. The recombinant CcEst1 expressed in Pichia pastoris acted on p-nitrophenyl acetate, α-naphthyl acetate, and methyl hydroxycinnamic acids, except for methyl sinapic acid. The enzyme released ferulic and acetic acids from wheat arabinoxylan and acetylated xylan respectively. Activity increased on the addition of endo-β-1,4-xylanase.     

Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain Trichoderma asperellum T-1

Trichoderma asperellum, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of Trichoderma reeseiQM6a, T. asperellum T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in T. asperellum T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in T. asperellum T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain T. asperellum T-1. All of the above results provided good references for better understanding T. asperellum T-1 and improving its application for lignocellulose degradation.     

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.