Arabinoxylan Arabinofuranohydrolase Research Articles

Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase-d3 and endo-xylanase activities and its application in the hydrolysis of cereal arabinoxylans.

Xylanolytic enzymes, with both endo-xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo-xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)-3-linked arabinofuranosyl residues of double-substituted xylopyranosyl units of arabinoxylan and arabinoxylan-derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo-oligosaccharides (XOSs) from beechwood xylan, xylohexaose and xylopentaose, indicating that rAbf43A exhibited endo-xylanase activity. Homology modelling based on AlphaFold2 and site-directed mutagenesis identified three non-catalytic residues (H161, A270 and L505) located in the substrate-binding pocket essential for its dual-functionality, while the mutation of A117 located in the -1 subsite to the proline residue only affected its endo-xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de-starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre-hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high-performance enzyme cocktails but also for investigating the structure-function relationship of GH43 multifunctional enzymes.

Taxonomic structure and function of the corn stover degradative microbial consortium GF‐20 following growth on different sources of nitrogen

AbstractMicrobial consortia have substantial promise to degrade returned straw biomass and implement conversion strategies for soil nutrients to improve the quality of cultivated land. However, the composition of microbial consortia under different nitrogen conditions, the interaction between members and the functions during the process of decomposition of lignocellulose remain poorly understood. This study comprehensively examines the functional potential and structural diversity of the GF‐20 microbial consortium that degrades lignocellulose and soil used for inoculation and will explore the associated metabolic pathway networks in Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) using metagenomic analyses. Analyses of the metagenomic taxonomic affiliation data showed that the soil used for inoculation primarily contained members from the phyla Proteobacteria, Bacteroidetes, Acidobacteria, Chloroflexi and Acidobacteria, as well as Proteobacteria and Bacteroidetes in microbial consortia (N1 and N6). However, N6 is likely to be able to reduce the diversity of microbial species and change the community structure in a more concentrated comparison with N1. Notably, the key microbes were determined to be Cellvibrio, Brevundimonas, Flavobacterium and Achromobacter, whose analogous physiological functions were critical for the degradation of lignocellulosic substrates. The COG annotation indicated that the microbial metabolic cluster was the predominant category in all the samples, while the metabolic genes in N6 were relatively less abundant compared with those in N1. The KEGG annotation demonstrated that the inoculation soil (Q) has more metabolic functions and pathways than those of the microbial consortia, and the number of orthologies, modules, pathways and enzymes in N6 was observably lower than that in N1. A more detailed analysis showed that both of the consortia had functional profiles that were highly similar. These functional profiles clearly differed compared with those in the Q community. The relative abundance (RA) of ABC transporters in the Q was higher than that in the microbial consortia and higher in N1 than in N6. However, the two‐component system of N1 and N6 was higher than that of Q because of their relation to the microbial degradation of lignocellulosic materials. Most of the abundances of lignocellulolytic enzymes in the Q were lower than those in the microbial consortia (N1 and N6), and the RAs of some of these enzymes (e.g., β‐glucosidase, xylan 1,4‐β‐xylosidase, arabinoxylan arabinofuranohydrolase, β‐galactosidase, feruloyl esterase, mannan endo‐1,4‐β‐mannosidase, alpha‐glucuronidase and arabinogalactan endo‐1,4‐β‐galactosidase) in N6 were significantly higher than those of N1.The taxonomic structure and function of the Q and that of the consortia differed significantly, and the use of urea resulted in a decrease in the taxonomic species of microorganisms. The changes in functional diversity were accompanied with variation in the microbial composition, many of which were related to the microbial degradation of lignocellulolytic materials. Most of the degradative lignocellulolytic function in Q was lower than those of the microbial consortia N1 and N6, which aid in the degradation of straw.

Comparative genome analysis of Bacillus velezensis reveals a potential for degrading lignocellulosic biomass.

Genomes of 24 sequenced Bacillus velezensis strains were characterized to identity shared and unique genes of lignocellulolytic enzymes and predict potential to degrade lignocellulose. All 24 strains had genes that encoded lignocellulolytic enzymes, with potential to degrade cellulose and hemicelluloses. Several lignocellulosic genes related to cellulose degradation were universally present, including one GH5 (endo-1,4-β-glucanase), one GH30 (glucan endo-1,6-β-glucosidase), two GH4 (6-phospho-β-glucosidase, 6-phospho-α-glucosidase), one GH1 (6-phospho-β-galactosidase), one GH16 (β-glucanase) and three GH32 (two sucrose-6-phosphate hydrolase and levanase). However, in the absence of gene(s) for cellobiohydrolase, it was predicted that none of the 24 strains would be able to directly hydrolyse cellulose. Regarding genes for hemicellulose degradation, four GH43 (1,4-β-xylosidase; except strain 9912D), one GH11 (endo-1,4-β-xylanase), three GH43 (two arabinan endo-1,5-α-L-arabinosidase and one arabinoxylan arabinofuranohydrolase), two GH51 (α-N-arabinofuranosidase), one GH30 (glucuronoxylanase), one GH26 (β-mannosidase) and one GH53 (arabinogalactan endo-1,4-β-galactosidase) were present. In addition, two PL1 (pectate lyase) and one PL9 (pectate lyase) with potential for pectin degradation were conserved among all 24 strains. In addition, all 24 Bacillus velezensis had limited representation of the auxiliary activities super-family, consistent with a limited ability to degrade lignin. Therefore, it was predicted that for these bacteria to degrade lignin, pretreatment of lignocellulosic substrates may be required. Finally, based on in silico studies, we inferred that Bacillus velezensis strains may degrade a range of polysaccharides in lignocellulosic biomasses.

Identification and characterization of GH62 bacterial α-l-arabinofuranosidase from thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched l-arabinofuranoses in wheat arabinoxylan.

We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity at pH 5.0 and 50 °C, and more than 70% of its initial enzymatic activity remained after incubation at pH 4.1-10.5, while SWUAbf62A lost all activity after 1 h at 60 °C. SWUAbf62A activity was stimulated by Ba2+, Ca2+, and Mn2+ and decreased by Ag+, Cu2+, Fe2+, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-l-arabinofuranoside or p-nitrophenyl-β-d-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg, and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using 1H-NMR analysis, SWUAbf62A was found to release l-arabinofuranoses singly linked to O-3 of wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase (XynSW3) and α-l-arabinofuranosidase, which releases arabinose linked to O-3 of double-substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and can be applied in various industrial biotechnologies.

Characterization of Ruminiclostridium josui arabinoxylan arabinofuranohydrolase, RjAxh43B, and RjAxh43B-containing xylanolytic complex.

A novel gene (axh43B) from Ruminiclostridium josui encoding a cellulosomal enzyme consisting of a catalytic module of subfamily GH43_10, a family-6 carbohydrate-binding module, and a dockerin module, was expressed using Escherichia coli. RjAxh43B released only arabinose from arabinoxylan and 23,33-di-α-l-arabinofuranosyl xylotriose, but not 32-α-l-arabinofuranosyl xylobiose or 23-α-l-arabinofuranosyl xylotriose, strongly suggesting that RjAxh43B is an arabinoxylan α-l-1,3-arabinofuranohydrolase capable of cleaving α-1,3-linked arabinose residues of doubly arabinosylated xylan. When Axh43B was mixed with the recombinant scaffolding protein RjCipA of R. josui at a molar ratio of 6:1, the activity of the RjAxh43B-RjCipA complex (6:1) toward insoluble wheat arabinoxylan was similar to that of RjAxh43B alone, suggesting that RjAxh43B does not show a proximity effect, which is defined as an activity enhancement effect caused by the presence of plural catalytic subunits adjoining each other. When RjAxh43A was mixed with xylanase RjXyn10C, they acted synergistically toward insoluble wheat arabinoxylan and rice straw powder in the absence of RjCipA. Furthermore, the RjAxh43B-RjXyn10C-RjCipA (3:3:3) complex had higher activity toward insoluble wheat arabinoxylan than a mixture of RjAxh43B and RjXyn10C without RjCipA, suggesting that incorporation of a xylanase and an α-l-arabinofuranosidase into a cellulosome is beneficial for more efficiently degrading arabinoxylan.

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited β-xylosidase activity toward a chromogenic substrate, p-nitrophenyl-β-d-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, α-l-Araf-(1→2)-d-Xylp or α-l-Araf-(1→3)-d-Xylp and α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.

Cell wall degradation is required for normal starch mobilisation in barley endosperm.

Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.

Metabolic potential of Bacillus subtilis 168 for the direct conversion of xylans to fermentation products.

Methylglucuronoxylans (MeGXn) and methylglucuronoarabinoxylans (MeGAXn) respectively comprise most of the hemicellulose fractions in dicots and monocots and, next to cellulose, are the major resources for the production of fuels and chemicals from lignocellulosics. With either MeGXn or MeGAXn as a substrate, Bacillus subtilis 168 accumulates acidic methylglucuronoxylotriose as a limit product following the uptake and metabolism of neutral xylooligosaccharides. Secreted GH11 endoxylanase (Xyn11A), GH30 endoxylanase (Xyn30C), and GH43 arabinoxylan arabinofuranohydrolase (Axh43) respectively encoded by the xynA, xynC, and xynD genes collectively contribute to the depolymerization of MeGAXn. Studies here demonstrate the complementary roles of these enzymes in the digestion of MeGAXn. Coordinate expression of the xynD and xynC genes defines an operon accounting for the Axh43-catalyzed release of arabinose followed by Xyn30C and Xyn11A-catalyzed depolymerization of MeGAXn. Both sources generate acetate and lactate as the principal fermentation products, with yields of 26% acetate and 32% lactate from MeGXn compared to 22% acetate and 21% lactate from MeGAXn. These studies of the GH43/GH30/GH11 system in B. subtilis 168 provide a basis for the further development of B. subtilis and related species as biocatalysts for direct conversion of hemicellulose derived from energy crops as well as agricultural and forest residues to chemical feedstocks.

A newly discovered arabinoxylan-specific arabinofuranohydrolase. Synergistic action with xylanases from different glycosyl hydrolase families.

Arabinofuranosidase Abf43A from Bacillussp. BP-7 i s a newly discovered arabinoxylan arabinofuranohydrolase (AXH). It is a modular enzyme comprised of a GH43 catalytic domain and a carbohydrate-binding module of family CBM6. Recombinant Abf43A showed high activity on arabinoxylans, being rye arabinoxylan the preferred substrate on which the purified enzyme exhibited a Km of 10.6 ± 3.3 mg/ml and a Vmax of 29.2 ± 3.4 U/mg. Thin-layer chromatography analysis of hydrolysis products showed arabinose as the only sugar released by the enzyme from its substrates. The GH43 and CBM6 modules of the enzyme were individually cloned and expressed in Escherichia coli. While the isolated catalytic GH43 module did not show hydrolytic activity, the purified CBM6 bound to soluble arabinoxylan in affinity gel electrophoresis analysis. Evaluation of cooperative activity of arabinofuranosidase Abf43A with xylanases from families GH10, GH11, andGH30, (Xyn10A, Xyn11E, and Xyn30D from Paenibacillus barcinonensis) on arabinoxylan depolymerization revealed that the studied enzyme showed synergism with Xyn11E, a 2.54-fold increase in the amount of sugars released. On the contrary, Abf43A did not show synergism with the xylanases of families GH10 or GH30 evaluated. The enzyme characterized contributes to understanding the role of this class of enzymes in the catalytic depolymerization of arabinoxylans and their potential for the production of valuable xylooligosaccharides from these abundant plant polymers.

Transcriptomic analysis of xylan utilization systems in Paenibacillus sp. strain JDR-2.

Xylans, including methylglucuronoxylans (MeGX(n)) and methylglucuronoarabinoxylans (MeGAXn), are the predominant polysaccharidesin hemicellulose fractions of dicots and monocots available for conversion to biofuels and chemicals. Paenibacillus sp. strain JDR-2 (Pjdr2) efficiently depolymerizes MeGX(n) and MeGAX(n) and assimilates the generated oligosaccharides, resulting in efficient saccharification and subsequent metabolism of these polysaccharides. A xylan utilization regulon encoding a cellassociated GH10 (glycoside hydrolase family 10) endoxylanase, transcriptional regulators, ABC (ATP binding cassette) transporters, an intracellular GH67 -glucuronidase, and other glycoside hydrolases contributes to complete metabolism. This GH10/GH67 system has been proposed to account for preferential utilization of xylans compared to free oligo- and monosaccharides. To identify additional genes contributing to MeGX(n) and MeGAXn utilization, the transcriptome of Pjdr2 has been sequenced following growth on each of these substrates as well as xylose and arabinose. Increased expression of genes with different substrates identified pathways common or unique to the utilization of MeGX(n) or MeGAX(n). Coordinate upregulation of genes comprising the GH10/GH67 xylan utilization regulon is accompanied with upregulation of genes encoding a GH11 endoxylanase and a GH115 -glucuronidase, providing evidence for a novel complementary pathway for processing xylans. Elevated expression of genes encoding a GH43 arabinoxylan arabinofuranohydrolase and an arabinose ABC transporter on MeGAX(n) but not on MeGX(n) supports a process in which arabinose may be removed extracellularly followed by its rapid assimilation.Further development of Pjdr2 for direct conversion of xylans to targeted products or introduction of these systems into fermentative strains of related bacteria may lead to biocatalysts for consolidated bioprocessing of hemicelluloses released from lignocellulose.

Purification and characterisation of arabinoxylan arabinofuranohydrolase I responsible for the filterability of barley malt

Dan’er is a widely grown malt barley cultivar in China, but its filterability defects have severely impeded its application in beer brewing. Previous investigations have suggested that we should identify the malt filterability correlated proteins, one of which was postulated to be arabinoxylan arabinofuranohydrolase I (AXAH-I). To verify this hypothesis, we purified AXAH-I from Dan’er malt, characterised its enzyme performance, and investigated its influence on filterability by adding different amounts of purified enzyme to the mash. With 6mUg−1 malt AXAH-I supplemented, the wort separation rate increased by 31.8%, viscosity decreased by 3.6%, and the endosperm reserve contents declined concomitantly. Unexpectedly, the wort turbidity increased with increasing AXAH-I. We also tried to optimise the use of currently available commercial enzyme products for filterability improvement in beer brewing, by supplementing them with purified AXAH-I and β-amylase. AXAH-I could be a functional component for novel commercial enzyme products in the beer industry.

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-d-glucurono-d-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-l-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.

Analysis of the arabinoxylan arabinofuranohydrolase gene family in barley does not support their involvement in the remodelling of endosperm cell walls during development

Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-β-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.

Life cycle expression analysis of three cell wall degradation-related genes in ethylene-treated grass

Ethylene regulates multiple developmental processes during a plant life cycle, but the effect of ethylene on the upregulation of senescence-, stress-, and post-harvest-related genes in forage grasses is poorly understood. In this work, we used quantitative PCR to determine whether ethylene application affected the expression of selected cell-wall degradation related genes that are typically upregulated post-harvest. The expression levels of beta-d-glucan exohydrolase isoenzyme, alpha glucosidase, and arabinoxylan arabinofuranohydrolase isoenzyme, all putative cell wall degrading enzymes, were quantified at six points in the life cycle of the model grass species Darnel ryegrass (Lolium temulentum L.). We also quantified the expression of ACC oxidase and ACC synthase in response to ethylene application to determine if endogenous upregulation of ethylene biosynthesis occurred. Grass developmental stage had a significant impact on gene expression response to ethylene-treatment, indicating that discrete life cycle stages present different ethylene-responsive windows for treatment. Under our experimental conditions, ACC oxidase and ACC synthase expression were downregulated in response to ethylene-treatment, suggesting that exogenous ethylene served an auto-inhibitory role. Transcripts corresponding to the three cell wall degradation related genes increased significantly in response to ethylene treatment, suggesting that ethylene may have future utility in the pretreatment of lignocellulosic biomass. To our knowledge, this is the first report of a life cycle analysis of ethylene-induced genes in forage grasses.

Identification of a GH62 α-l-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum

An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a "Glyco_hydro_62" was present at position 28-299 of AXS5. This is a family of α-L-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. (1)H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding D-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.

Domain analysis of a modular alpha-L-Arabinofuranosidase with a unique carbohydrate binding strategy from the fiber-degrading bacterium Fibrobacter succinogenes S85.

Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k(cat) for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the K(m) for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with beta-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.

Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family

AXHs (arabinoxylan arabinofuranohydrolases) are alpha-L-arabinofuranosidases that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues of arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues: BsAXH-m2,3 (B. subtilis AXH-m2,3). Crystallographic analysis reveals a two-domain structure for this enzyme: a catalytic domain displaying a five-bladed beta-propeller fold characteristic of GH (glycoside hydrolase) family 43 and a CBM (carbohydrate-binding module) with a beta-sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic stacking interactions, which probably allow the positional flexibility needed to hydrolyse both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, beta-xylosidase and alpha-L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone.

Remodelling of arabinoxylan in wheat (Triticum aestivum) endosperm cell walls during grain filling

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393-1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. (1)H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by (1)H NMR.

Crystallization and preliminary X-ray analysis of an arabinoxylan arabinofuranohydrolase fromBacillus subtilis

Arabinoxylan arabinofuranohydrolases (AXH) are alpha-L-arabinofuranosidases (EC 3.2.1.55) that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl residues from arabinoxylan, hence their name. In this study, the crystallization and preliminary X-ray analysis of the AXH from Bacillus subtilis, a glycoside hydrolase belonging to family 43, is described. Purified recombinant AXH crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.7, b = 73.7, c = 106.5 A. X-ray diffraction data were collected to a resolution of 1.55 A.