Acetyl Xylan Research Articles

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.

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.

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.

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.

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).

Effects of hemicelluloses on dehydrogenative polymerization of monolignols with cationic cell wall-bound peroxidase

To elucidate the influence of polysaccharides on hardwood lignification, dehydrogenative polymerization of monolignols, coniferyl alcohol (CA) and sinapyl alcohol (SA), was attempted with recombinant cationic cell wall-bound peroxidase (rCWPO-C) and horseradish peroxidase (HRP) in measurement cells of a quartz crystal microbalance with dissipation (QCM-D). Hardwood cellulose nanofibers were anchored; hemicelluloses, xylan, partially acetylated xylan (AcXY), galactoglucomannan, and xyloglucan, and the enzymes were subsequently adsorbed onto the QCM-D sensor surface, enabling fabrication of artificial polysaccharide matrices. The largest amount of rCWPO-C is found to be adsorbed onto AcXY among all the polysaccharides, which affords the largest amount and size of spherical dehydrogenation polymers (DHPs) from both CA and SA. In contrast, no DHP and a small amount of DHPs are formed from SA and CA, respectively, by HRP catalysis in all of the polysaccharide matrices. This study demonstrates important functions of a real tree-derived peroxidase, rCWPO-C, and AcXY for hardwood lignification.

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.

Arabidopsis GELP7 functions as a plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency.

Acetyl substitution on the xylan chain is critical for stable interaction with cellulose and other cell wall polymers in the secondary cell wall. Xylan acetylation pattern is governed by Golgi and extracellular localized acetyl xylan esterase (AXE). We investigated the role of Arabidopsis clade Id from the GDSL esterase/lipase or GELP family in polysaccharide deacetylation. The investigation of the AtGELP7 T-DNA mutant line showed a decrease in stem esterase activity and an increase in stem acetyl content. We further generated overexpressor AtGELP7 transgenic lines, and these lines showed an increase in AXE activity and a decrease in xylan acetylation compared to wild-type plants. Therefore, we have named this enzyme as AtAXE1. The subcellular localization and immunoblot studies showed that the AtAXE1 enzyme is secreted out, associated with the plasma membrane and involved in xylan de-esterification post-synthesis. The cellulose digestibility was improved in AtAXE1 overexpressor lines without pre-treatment, after alkali and xylanases pre-treatment. Furthermore, we have also established that the AtGELP7 gene is upregulated in the overexpressor line of AtMYB46, a secondary cell wall specific transcription factor. This transcriptional regulation can drive AtGELP7 or AtAXE1 to perform de-esterification of xylan in a tissue-specific manner. Overall, these data suggest that AtGELP7 overexpression in Arabidopsis reduces xylan acetylation and improves digestibility properties of polysaccharides of stem lignocellulosic biomass.

Characterisation of a Novel Acetyl Xylan Esterase (BaAXE) Screened from the Gut Microbiota of the Common Black Slug (Arion ater).

Acetyl xylan esterases (AXEs) are enzymes capable of hydrolysing the acetyl bonds in acetylated xylan, allowing for enhanced activity of backbone-depolymerizing enzymes. Bioprospecting novel AXE is essential in designing enzyme cocktails with desired characteristics targeting the complete breakdown of lignocellulose. In this article, we report the characterisation of a novel AXE identified as Gene_id_40363 in the metagenomic library analysed from the gut microbiota of the common black slug. The conserved domain description was identified with an NCBI BLASTp search using the translated nucleotide sequence as a query. The activity of the recombinant enzyme was tested on various synthetic substrates and acetylated substrates. The protein sequence matched the conserved domain described as putative hydrolase and aligned closely to an uncharacterized esterase from Buttiauxella agrestis, hence the designation as BaAXE. BaAXE showed low sequence similarity among characterized CE family proteins with an available 3D structure. BaAXE was active on 4-nitrophenyl acetate, reporting a specific activity of 78.12 U/mg and a Km value of 0.43 mM. The enzyme showed optimal activity at 40 °C and pH 8 and showed high thermal stability, retaining over 40% activity after 2 h of incubation from 40 °C to 100 °C. BaAXE hydrolysed acetyl bonds, releasing acetic acid from acetylated xylan and β-D-glucose pentaacetate. BaAXE has great potential for biotechnological applications harnessing its unique characteristics. In addition, this proves the possibility of bioprospecting novel enzymes from understudied environments.

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans.

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases.

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.

Xylan Deconstruction by Thermophilic Thermoanaerobacterium bryantii Hemicellulases Is Stimulated by Two Oxidoreductases

Thermoanaerobacterium bryantii strain mel9T is a thermophilic bacterium isolated from a waste pile of a corn-canning factory. The genome of T. bryantii mel9T was sequenced and a hemicellulase gene cluster was identified. The cluster encodes seven putative enzymes, which are likely an endoxylanase, an α-glucuronidase, two oxidoreductases, two β-xylosidases, and one acetyl xylan esterase. These genes were designated tbxyn10A, tbagu67A, tbheoA, tbheoB, tbxyl52A, tbxyl39A, and tbaxe1A, respectively. Only TbXyn10A released reducing sugars from birchwood xylan, as shown by thin-layer chromatography analysis. The five components of the hemicellulase cluster (TbXyn10A, TbXyl39A, TbXyl52A, TbAgu67A, and TbAxe1A) functioned in synergy to hydrolyze birchwood xylan. Surprisingly, the two putative oxidoreductases increased the enzymatic activities of the gene products from the xylanolytic gene cluster in the presence of NADH and manganese ions. The two oxidoreductases were therefore named Hemicellulase-Enhancing Oxidoreductases (HEOs). All seven enzymes were thermophilic and acted in synergy to degrade xylans at 60 °C. Except for TbXyn10A, the other enzymes encoded by the gene cluster were conserved with high amino acid identities (85–100%) in three other Thermoanaerobacterium species. The conservation of the gene cluster is, therefore, suggestive of an important role of these enzymes in xylan degradation by these bacteria. The mechanism for enhancement of hemicellulose degradation by the HEOs is under investigation. It is anticipated, however, that the discovery of these new actors in hemicellulose deconstruction will have a significant impact on plant cell wall deconstruction in the biofuel industry.

A new strategy to improve ramie degumming based on removal of the xylan branched structure

Ramie fiber is known as the “king of natural fibers,” and the key to its wide application is efficient and green manufacturing. Microbial degumming has gradually become a hot area of research due to its environmental protection and mild operating conditions. However, some gummy materials remain after microbial degumming. Xylan is the main component of residual gums; its acetylated branched chains create the space barrier that makes the removal of hemicellulose difficult during ramie degumming. An acetyl xylan esterase (AXE) was obtained from Bacillus pumilus and characterized to solve this problem. Its optimum temperature and pH were 35°C and 8.0, respectively, and it had good temperature and pH stability. These properties were consistent with the conditions of ramie degumming and they laid a foundation for the application of AXE in ramie degumming. Besides, an engineered strain with a high activity of AXE was constructed successfully on the basis of the wild-type degumming strain Pectobacterium carotovorum HG-49 and used for ramie degumming. The removal rate of hemicellulose and total gums by the engineered strain increased by 4.89% and 2.53%, respectively, compared with that of the wild-type strain. Moreover, the role of this AXE in ramie degumming was further proven by X-ray diffraction and scanning electron microscopy. This study showed that AXE played an important role in the removal of hemicellulose in the degumming process of ramie fibers, thus providing a promising degumming strategy for ramie and other bast fiber plants.

Saccharification Potential of Transgenic Greenhouse- and Field-Grown Aspen Engineered for Reduced Xylan Acetylation.

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.

Xylanolytic enzyme consortia from Bacillus sp. NIORKP76 for improved biobleaching of kraft pulp.

A cellulase-free xylanolytic enzyme consortia consisting of a xylanase, arabinofuranosidase, and acetyl xylan esterase produced by Bacillus sp. NIORKP76 isolate under solid-state fermentation was assessed for its bio-bleaching ability on kraft pulp. In the biobleaching analysis, the xylanase dose of 5Ug-1 dry pulp denoted the optimum bleaching of pulp at 40°C and pH 8.0 after 2h of treatment. The reduction in kappa number of pre-treated hardwood pulp using xylanolytic enzyme consortium (XEC) was found to be ~ 55%, while solo xylanase could reduce the kappa number to 44-46%. In the case of chemical bagasse pulp, a reduction of ~ 27.5% and 19-20% was seen in kappa number using XEC and solo xylanase, respectively. Enzyme-treated pulp (HW and CB) showed a 50% reduction in hypochlorite consumption during the chlorine treatment. The current study results reveal the significant potential of xylanolytic enzyme consortium from Bacillus sp. NIORKP76 on the environmentally friendly bio-bleaching process.

Identification and characterization of an acetyl xylan esterase from Aspergillus oryzae

In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillusoryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with α-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50°C, respectively, and was stable up to 50°C. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of α-naphthyl esters (C2-C16), was α-naphthyl propionate (C3), with an activity of 0.35 ± 0.006 units/mg protein. No significant difference of the Km value was observed between C3 (2.3 ± 0.7mM) and C2 (1.9 ± 0.4mM). In contrast, kcat value for C3 (18 ± 3.9 s-1) was higher compared to C2 (4.5 ± 0.7 s-1). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.

Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass

BackgroundXylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the β-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis.ResultsxynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50–55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold.ConclusionThe supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.