Acetyl Xylan Esterase Research Articles

Acetylxylan esterase is the key to the host specialization of wood-decay fungi predicted by random forest machine-learning algorithm

Wood-decay fungi produce extracellular enzymes that metabolize wood components such as cellulose, hemicellulose and lignin. Each fungus has a preference of wood species as the host, but identification of these preferences requires a huge amount of cultivation data. Here, we developed a method of predicting the wood species preference, Angiosperm specialist or Gymnosperm specialist or generalist, of wood-decay fungi using the random forest machine-learning algorithm, trained on the numbers of families associated with host specialization in the Carbohydrate-Active enZymes database. The accuracy of the prediction was about 80%, which is lower than that of the classification of white- and brown-rot fungi (more than 98%) by the same method, but the reason for this may be the ambiguity of the definition of “preference” and “generalists”. Carbohydrate esterase (CE) family 1 acetylxylan esterase was the most significant contributor to the prediction of host specialization, followed by family 1 carbohydrate-binding module and CE family 15, mainly containing glucuronoyl esterases. These results suggest that the ability to degrade glucuronoacetylxylan, a major hemicellulose of Angiosperm, is the key factor determining the host specialization of wood-decay fungi.

Homologous expression, purification, and characterization of a recombinant acetylxylan esterase from Aspergillus nidulans

Acetylxylan esterases (AXEs) are essential enzymes that break down the acetyl groups in acetylated xylan found in plant cell walls polysaccharides. They work synergistically with backbone-depolymerizing xylanolytic enzymes to accelerate the degradation of complex polysaccharides. In this study, we cloned the gene axeA, which encodes the acetylxylan esterase from Aspergillus nidulans FGSC A4 (AxeAN), into the pEXPYR expression vector and introduced it into the high protein-producing strain A. nidulans A773. The purified AxeAN, with a molecular weight of 33.5 kDa as confirmed by SDS-PAGE, was found to be active on ρ-nitrophenyl acetate (ρNPA), exhibiting a remarkably high specific activity (170 U mg−1) at pH 7.0 and 55 °C. AxeAN demonstrated stability over a wide pH range (5.5–9.0), retaining >80% of its initial activity after 24 h. The KM and Vmax were 0.098 mmol L−1 and 320 U mg−1, respectively, using ρNPA as a substrate. We also evaluated the synergistic effect of AxeAN with an endo-1,4-β-xylanase from Malbranchea pulchella (MpXyn10) in the hydrolysis of four different xylans (Birchwood, Beechwood, Oat spelt, and Arabinoxylan) to produce xylooligosaccharides (XOS). The best results were obtained using Birchwood xylan as substrate and MpXyn10-AxeAN as biocatalysts after 24 h of reaction (50 °C), with a XOS-yield of 91%, value 41% higher when compared to MpXyn10 (XOS-yield of 63%). These findings showed the potential of the application of AxeAN, together with other xylanases, to produce xylooligosaccharides with high purity and other products with high added value in the field of lignocellulosic biorefinery.

Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L−1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8–25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

Enhancing Paenibacillus sp. Cold-Active Acetyl Xylan Esterase Activity through Semi-Rational Protein Engineering

Interest in protein engineering for the enzymatic production of valuable products, such as pharmaceutical compounds and biofuels, is growing rapidly. The cold-active acetyl xylan esterase from Paenibacillus sp. (PbAcE) presents unusually broad substrate specificity. Here, we engineered a hydrophobic substrate-binding pocket to enable the accommodation of relatively large alcohol substrates, such as linalyl acetate and α-terpinyl acetate. To identify candidate residues for engineering, we performed covalent docking of substrates to the Ser185 active site using the HCovDock program. Functional hotspots were analyzed using HotSpot Wizard 3.1. Lys91, His93, and Tyr182 were selected for site-saturation mutagenesis (SSM). After generating the SSM mutant library, a qualitative colorimetric assay was conducted to identify positive mutants. Three, two, and five single mutants were selected for Lys91, His93, and Tyr182, respectively. The best single mutants were then sequentially combined to generate double and triple mutants. Single mutants exhibited a 10–30% increase in activity compared to that of wild-type PbAcE, while no significant synergistic improvements were observed in the double and triple mutants. The increase in activity against both linalyl acetate and α-terpinyl acetate was similar. Mutation did not affect the acetyl binding and catalysis. Further research on the acetyl binding pocket will provide insights into substrate specificity and aid in efficient biocatalyst development for industrial applications.

Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity.

Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58s-1mM-1, 1.09s-1mM-1, and 0.062s-1mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20min at 70°C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility

BackgroundLignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.ResultsArabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.ConclusionsSimultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Characterization of a novel multifunctional β-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea

Herein, a novel multifunctional enzyme β-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of β-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of β-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

Production of acetic acid from wheat bran by catalysis of an acetoxylan esterase

In this study, a gene encoding for acetylxylan esterase was cloned and expressed in E. coli. A single uniform band with molecular weight of 31.2 kDa was observed in SDS-PAGE electrophoresis. Served as the substrate, p-nitrophenol butyrate was employed to detect the recombinant enzyme activity. It exhibited activity at a wide temperature range (30–100 °C) and pH (5.0–9.0) with the optimal temperature of 70 °C and pH 8.0. Acetylxylan esterase showed two substrates’ specificities with the highest Vmax of 177.2 U/mg and Km of 20.98 mM against p-nitrophenol butyrate. Meanwhile, the Vmax of p-nitrophenol acetate was 137.0 U/mg and Km 12.16 mM. The acetic acid yield of 0.39 g/g was obtained (70 °C and pH 8.0) from wheat bran pretreated using amylase and papain. This study showed the highest yield up to date and developed a promising strategy for acetic acid production using wheat bran.

Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in Escherichia coli.

The strategy of active aggregation tag fusion expression with target proteins can solve the problems of restricted expression, inefficient purification, and laborious immobilization faced in the production of recombinant proteins in Escherichia coli. We localized a novel active aggregation peptide HlyA60 from the hemolysin A secretion system, which can effectively induce aggregate formation with satisfactory protein activities in E. coli after fusion expression with the protein of interest. Based on structural prediction and surface properties, the process of active aggregation of HlyA60 through electrostatic interactions and hydrophobic interactions was analyzed. To investigate the potential application of HlyA60 as an efficient aggregation tag, it was fused with acetyl xylan esterase and lipase A, separately. The resulting fusion proteins demonstrated active aggregation rates of 97.6 and 66.7%, respectively, leading to 1.9-fold and 1.7-fold increases in bacterial density at the end of fermentation. The AXE-HlyA60 fusion protein, which exhibited superior performance, was subjected to purification and immobilization. It was able to achieve column-free purification with an impressive 98.8% recovery and in situ immobilization; the immobilization enabled 30 cycles of reactions to take place with 85% residual activity maintained. Our findings provide a novel tool for efficiently producing recombinant proteins in E. coli.

The role of CE16 exo-deacetylases in hemicellulolytic enzyme mixtures revealed by the biochemical and structural study of the novel TtCE16B esterase

Acetyl esterases belonging to the carbohydrate esterase family 16 (CE16) is a growing group of enzymes, with exceptional diversity regarding substrate specificity and regioselectivity. However, further insight into the CE16 specificity is required for their efficient biotechnological exploitation. In this work, exo-deacetylase TtCE16B from Thermothelomyces thermophila was heterologously expressed and biochemically characterized. The esterase targets positions O-3 and O-4 of singly and doubly acetylated non-reducing-end xylopyranosyl residues, provided the presence of a free vicinal hydroxyl group at position O-4 and O-3, respectively. Crystal structure of TtCE16B, the first representative among the CE16 enzymes, in apo- and product-bound form, allowed the identification of residues forming the catalytic triad and oxyanion hole, as well as the structural elements related to the enzyme preference for oligomers. The role of TtCE16B in hemicellulose degradation was investigated on acetylated xylan from birchwood and pre-treated beechwood biomass. TtCE16B exhibited complementary activity to commercially available OCE6 acetylxylan esterase. Moreover, it showed synergistic effects with SrXyl43 β-xylosidase. Overall, supplementation of xylan-targeting enzymatic mixtures with both TtCE16B and OCE6 esterases led to a 3-fold or 4-fold increase in xylose release, when using TmXyn10 and TtXyn30A xylanases respectively.

Enhanced Activity and Stability of an Acetyl Xylan Esterase in Hydrophilic Alcohols through Site-Directed Mutagenesis.

Current demands for the development of suitable biocatalysts showing high process performance is stimulated by the need to replace current chemical synthesis with cleaner alternatives. A drawback to the use of biocatalysts for unique applications is their low performance in industrial conditions. Hence, enzymes with improved performance are needed to achieve innovative and sustainable biocatalysis. In this study, we report the improved performance of an engineered acetyl xylan esterase (BaAXE) in a hydrophilic organic solvent. The structure of BaAXE was partitioned into a substrate-binding region and a solvent-affecting region. Using a rational design approach, charged residues were introduced at protein surfaces in the solvent-affecting region. Two sites present in the solvent-affecting region, A12D and Q143E, were selected for site-directed mutagenesis, which generated the mutants MUT12, MUT143 and MUT12-143. The mutants MUT12 and MUT143 reported lower Km (0.29 mM and 0.27 mM, respectively) compared to the wildtype (0.41 mM). The performance of the mutants in organic solvents was assessed after enzyme incubation in various strengths of alcohols. The mutants showed improved activity and stability compared to the wild type in low strengths of ethanol and methanol. However, the activity of MUT143 was lost in 40% methanol while MUT12 and MUT12-143 retained over 70% residual activity in this environment. Computational analysis links the improved performance of MUT12 and MUT12-143 to novel intermolecular interactions that are absent in MUT143. This work supports the rationale for protein engineering to augment the characteristics of wild-type proteins and provides more insight into the role of charged residues in conferring stability.

Identification of a Coprinellus strain and its application in Eucommia ulmoides gum extraction by fermenting leaves.

White rot fungi is a kind of filamentous fungi which can degrade lignin, hemicellulose and cellulose effectively. In this study, a wild white rot fungi collected from Pingba Town, Bijie City of China was identified as Coprinellus disseminatus (fruiting body) based on morphological and molecular identification. The mycelium of C. disseminatus cultured in the medium supplemented xylan as carbon showed the higher xylanase (XLE) and cellulase (CLE) activity. Further, the activities of tissue degradation-related enzymes including XLE, CLE, acetyl xylanesterase (AXE) and α-L-arabinofuran glycosidase (α-L-AF) were determined after fermenting Eucommia ulmoides leaves by inoculating C. disseminatus mycelium. The results showed that the activities of XLE, CLE, AXE and α-L-AF of mycelium cultured in xylan-contained medium reached the maximum at 5 d after inoculation, which were 777.606 ± 4.248UmL-1, 9.594 ± 0.008UmL-1, 4.567 ± 0.026UmL-1 and 3.497 ± 0.10UmL-1 respectively. Also, the activities of AXE and α-L-AF both reached the maximum in C. disseminatus mycelium cultured in glucose-contained medium. By comparing the yield of E. ulmoides gum under different fermentation treatments, the extraction yield of E. ulmoides gum were 2.156 ± 0.031% and 2.142 ± 0.044% at 7 d and 14 d after fermentation with mycelium supplemented xylan as carbon source, which were significantly higher than other groups. This study provides a theoretical reference for the preparation of E. ulmoides gum by large-scale fermentation of E. ulmoides leaves with C. disseminatus.

Characterization of a novel carbohydrate esterase 7 family acetyl xylan esterase from Thermobifida halotolerans YIM 90462T

The newly semi-synthetic β-lactam antibiotics play critical roles in prevention and treatment of β-lactam antibiotic-resistant bacteria. Acetyl xylan esterases belong to carbohydrate esterase 7 family can specifically hydrolyze 7-aminocephalosporanic acid (7-ACA) to produce deacetyl-7-aminocephalosporanic acid (D-7ACA) which is one of the most important starting materials to synthesize newly semi-synthetic β-lactam antibiotics. Here, an effective acetyl xylan esterase, ThAXE, from Thermobifida halotolerans YIM 90462 T, was firstly identified and characterized. ThAXE exhibited its highest catalytic activity against p-nitrophenyl acetate (p-NPC2) at pH 8.5 and 50 °C, with Km and Kcat/Km were 1.0 ± 0.1 mM and 2262.9 mM−1 s−1, respectively. Additionally, ThAXE can hydrolyze the acetyl group of 7-ACA at its C3' position with the specific activity of 7.4 U mg−1. Furthermore, molecular docking combined with site-directed mutagenesis validation demonstrated that the pocket of catalytic residues Ser186, Asp272, and His301 could accommodate p-NPC2 and 7-ACA ligands. This is the first report of a novel acetyl xylan esterase from T. halotolerans that has considerable potential biotechnological values.

Production of Designer Xylose-Acetic Acid Enriched Hydrolysate from Bioenergy Sorghum, Oilcane, and Energycane Bagasses

Xylan accounts for up to 40% of the structural carbohydrates in lignocellulosic feedstocks. Along with xylan, acetic acid in sources of hemicellulose can be recovered and marketed as a commodity chemical. Through vibrant bioprocessing innovations, converting xylose and acetic acid into high-value bioproducts via microbial cultures improves the feasibility of lignocellulosic biorefineries. Enzymatic hydrolysis using xylanase supplemented with acetylxylan esterase (AXE) was applied to prepare xylose-acetic acid enriched hydrolysates from bioenergy sorghum, oilcane, or energycane using sequential hydrothermal-mechanical pretreatment. Various biomass solids contents (15 to 25%, w/v) and xylanase loadings (140 to 280 FXU/g biomass) were tested to maximize xylose and acetic acid titers. The xylose and acetic acid yields were significantly improved by supplementing with AXE. The optimal yields of xylose and acetic acid were 92.29% and 62.26% obtained from hydrolyzing energycane and oilcane at 25% and 15% w/v biomass solids using 280 FXU xylanase/g biomass and AXE, respectively.

Deacetylation of Arabinofuranosylated Xylopyranosyl Residues Related to Plant Xylan: Significant Differences Between Xylan Deacetylases Classified into Various Carbohydrate Esterase Families.

A chemical synthesis of two novel phenyl glycosides of trisaccharides related to acetylarabinoxylan is described. The trisaccharides bear acetyl and arabinofuranosyl moieties at the non-reducing-end xylopyranosyl residue, which is substituted at positions 2 and 3. Both compounds were treated with various xylan deacetylases classified in different carbohydrate esterase (CE) families and significant differences between the families were found. While the arabinosylation hampers deacetylation by CE2-CE5 and CE12 family members, both epitopes are deesterified by CE1 and in particular CE6 enzymes. The 3-O-acetylated 2-O-arabinosylated compound is also processed by CE7 and majority of CE16 esterases, but not by a hitherto non-classified Flavobacterium johnsoniae acetylxylan esterase. The data suggests that a slow deesterification of the 2-O-acetylated 3-O-arabinosylated compound may be due to the acetyl group migration followed by deacetylation of this migration product.

Comparative genomic and transcriptome analysis of Bacillus velezensis CL-4 fermented corn germ meal

Bacillus, an excellent organic-degrading agent, can degrade lignocellulose. Notably, some B. velezensis strains encode lignocellulases. However, their ability to degrade lignocellulose in fermented feed is not much appreciated. This study performed a comparative genomic analysis of twenty-three B. velezensis strains to find common carbohydrate-active enzymes (CAZymes) encoding genes and evaluated their potential to degrade lignocellulose. The comparative genomic and CAZyme database-based analyses identified several potential CAZymes genes that degrade cellulose (GH1, GH4, GH5, GH13, GH16, GH32, PL1, and PL9), hemicellulose (GH11, GH26, GH43, GH51, and CE3) and lignin (AA4, AA6, AA7, and AA10). Furthermore, Illumina RNA-seq transcriptome analysis revealed the expression of more than 1794 genes in B. velezensis CL-4 fermented corn germ meal at 48 h (FCGM 48 h). Gene ontology analysis of expressed genes revealed their enrichment in hydrolase activity (breaking the glycosyl bonds during carbohydrate metabolism), indicating the upregulation of CAZymes. In total, 58 differentially upregulated CAZymes-encoding genes were identified in FCGM 48 h compared to FCGM 0 h. The upregulated CAZymes-encoding genes were related to cellulose (6-phospho-β-galactosidase and 6-phospho-α-glucosidase), starch (α-glucosidase and α-amylase), pectin (pectin lyase), and hemicellulose (arabinan endo-1,5-α-L-arabinosidase, xylan 1,4-beta-xylosidase, α-N-arabinofuranosidase, and acetyl xylan esterase). Importantly, arabinoxylan degradation mainly occurred in FCGM 48 h, followed by partial degradation of cellulose, pectin, and starch. This study can support the development of enzymatic cocktails for the solid-state fermented feed (SFF).

Transcriptomic analysis of lignocellulose degradation by Streptomyces coelicolor A3(2) and elicitation of secondary metabolites production.

Streptomyces coelicolor A3(2) is considered as the model strain among the Streptomyces and has the capacity to produce several natural molecules. Our hypothesis was that cultivation of the strain onto a complex carbon source such as wheat bran (WB) would induce the production of various secondary metabolites due to the presence of complex polysaccharides. A multiapproach has been performed in order to investigate: (1) whether that strain could degrade lignocellulose; (2) which enzymatic and metabolic pathways secondary were over-expressed when grown on WB. The transcriptomic approach showed the expression of several CAZymes significantly expressed when grown on WB such as endoglucanases (encoding for GH74, GH5_8, and GH12) and xylanases (GH11 and CE4 encoding for respectively endo-1,4-beta-xylanase and an acetyl-xylan esterase). Enzymatic activities showed an expression of xylanase (115.3±32.2 mUI/ml) and laccase-peroxidase (101.5±10.9 mUI/ml) during WB degradation by S. coelicolor A3(2). Metabolomics showed that the production of secondary metabolites differed between growth on either glucose or WB as carbon source, which may be correlated to the complexity of carbon compounds within WB, which are similar to the ones encountered in soils and should represent more the in situ carbon conditions which Streptomyces might face off. This opens opportunities for the bioproduction of molecules of interest from WB.

Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study

Acetylxylan esterase plays a crucial role in xylan hydrolysis as the acetyl side-groups restrict endoxylanase action by stearic hindrance. In this study, an acetylxylan esterase (AXE-HAS10: 960 bp & 319 a.a) putative ORF from Halalkalibacterium halodurans NAH-Egypt was extensively studied through heterologous overexpression in Escherichia coli, biochemical characterization, and structural modeling. The AXE-HAS10 tertiary structure was predicted by the Local Meta Threading Server. AXE-HAS10 belongs to the carbohydrate esterase Family 7. Purified to homogeneity AXE-HAS10 showed specific activity (36.99 U/mg), fold purification (11.42), and molecular mass (41.39 kDa). AXE-HAS10 showed optimal pH (8.5) and temperature (40 oC). After 15 h of incubation at pH 7.0–9.0, AXE-HAS10 maintained 100% activity. After 120 min at 35 and 40 oC, the retained activity was 80 and 50%, respectively. At 10 mM Mn2+, Fe3+, K+, and Ca2+ after 30 min, retained activity was 329 ± 15, 212 ± 5.2, 123 ± 1.4, and 120 ± 3.0%, respectively. After 30 min of preincubation with triton x-100, SDS, and CTAB at 0.1% (v/v), the retained activity was 150 ± 19, 88 ± 4, and 82 ± 7%, respectively. At 6.0 M NaCl after 30 min, retained activity was 58%. A 1.44-fold enhancement of beechwood xylan hydrolysis was achieved by AXE-HAS10 and Penicillium chrysogenum DSM105774 β-xylanase concurrently. Present data underpins AXE-HAS10 as a promising AXE for industrial exploitation.

Functional analysis of the N-terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N‐terminal region (NTR; residues 23–135) between the signal sequence (residues 1–22) and the catalytic domain (residues 136–324), which is of unknown function. Our previous study revealed the crystal structure of the wild‐type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared with that of the WT. The NTR‐truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable with those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.

Acetylated Xylan Degradation by Glycoside Hydrolase Family 10 and 11 Xylanases from the White-rot Fungus <i>Phanerochaete chrysosporium</i>

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate-Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase (CE) family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply co-evolution of GH11 xylanases and CE1 AXEs.