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

BACKGROUND: 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. METHODS AND RESULTS: 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 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-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 20 min 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/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: 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.

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.

Active site architecture of an acetyl xylan esterase indicates a novel cold adaptation strategy.

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.

A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases.

The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multigene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate-binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs and cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment.

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.

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 ß-(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.

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 ß-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.

Developing a Library of Mannose-Based Mono- and Disaccharides: A General Chemoenzymatic Approach to Monohydroxylated Building Blocks.

Regioselective deprotection of acetylated mannose-based mono- and disaccharides differently functionalized in anomeric position was achieved by enzymatic hydrolysis. Candida rugosa lipase (CRL) and Bacillus pumilus acetyl xylan esterase (AXE) were immobilized on octyl-Sepharose and glyoxyl-agarose, respectively. The regioselectivity of the biocatalysts was affected by the sugar structure and functionalization in anomeric position. Generally, CRL was able to catalyze regioselective deprotection of acetylated monosaccharides in C6 position. When acetylated disaccharides were used as substrates, AXE exhibited a marked preference for the C2, or C6 position when C2 was involved in the glycosidic bond. By selecting the best enzyme for each substrate in terms of activity and regioselectivity, we prepared a small library of differently monohydroxylated building blocks that could be used as intermediates for the synthesis of mannosylated glycoconjugate vaccines targeting mannose receptors of antigen presenting cells.

Characterization of an acetyl xylan esterase from the marine bacterium Ochrovirga pacifica and its synergism with xylanase on beechwood xylan.

BACKGROUND: Acetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica. RESULTS: The AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold. CONCLUSION: Resulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates.

Designer bioemulsifiers based on combinations of different polysaccharides with the novel emulsifying esterase AXE from Bacillus subtilis CICC 20034.

BACKGROUND: Bioemulsifiers are surface-active compounds, which exhibit advantages including low toxicity, higher biodegradability and biocompatibility over synthetic chemical surfactants. Despite their potential benefits, some obstacles impede the practical applications of bioemulsifiers, including low yields and high purification costs. Here, we aimed to exploit a novel protein bioemulsifier with efficient emulsifying activity and low-production cost, as well as proposed a design-bioemulsifier system that meets different requirements of industrial emulsification in the most economical way. RESULTS: The esterase AXE was first reported for its efficient emulsifying activity and had been studied for possible application as a protein bioemulsifier. AXE showed an excellent emulsification effect with different hydrophobic substrates, especially short-chain aliphatic and benzene derivatives, as well as excellent stability under extreme conditions such as high temperature (85 °C) and acidic conditions. AXE also exhibited good stability over a range of NaCl, MgSO4, and CaCl2 concentrations from 0 to 1000 mM, and the emulsifying activity even showed a slight increase at salt concentrations over 500 mM. A design-bioemulsifier system was proposed that uses AXE in combination with a variety of polysaccharides to form efficient bioemulsifier, which enhanced the emulsifying activity and further lowered the concentration of AXE needed in the complex. CONCLUSIONS: AXE showed a great application potential as a novel bioemulsifier with excellent emulsifying ability. The AXE-based-designer bioemulsifier could be obtained in the most economical way and open broad new fields for low-cost, environmentally friendly bioemulsifiers.

Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family.

A hot desert hypolith metagenomic DNA sequence data set was screened in silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1NaM1). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg-1 and 3.26 × 106 M-1 s-1, respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2.03 Å, and a comparison with the structures and models of more thermostable carbohydrate esterase 7 (CE7) family enzymes and variants of NaM1 from a directed evolution experiment suggests that reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S) not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency but also increased the acyl moiety substrate range of NaM1.IMPORTANCE AcXEs belong to nine carbohydrate esterase families (CE1 to CE7, CE12, and CE16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterized members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1NaM1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone PCR and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other α/ß hydrolase enzymes.

A novel trifunctional, family GH10 enzyme from Acidothermus cellulolyticus 11B, exhibiting endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities.

A novel, family GH10 enzyme, Xyn10B from Acidothermus cellulolyticus 11B was cloned and expressed in Escherichia coli. This enzyme was purified to homogeneity by binding to regenerated amorphous cellulose. It had higher binding on Avicel as compared to insoluble xylan due to the presence of cellulose-binding domains, CBM3 and CBM2. This enzyme was optimally active at 70 °C and pH 6.0. It was stable up to 70 °C while the CD spectroscopy analysis showed thermal unfolding at 80 °C. Xyn10B was found to be a trifunctional enzyme having endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities. Its activities against beechwood xylan, p-Nitrophenyl arabinofuranoside and p-Nitrophenyl acetate were found to be 126,480, 10,350 and 17,250 U µmol-1, respectively. Xyn10B was highly active producing xylobiose and xylose as the major end products, as well as debranching the substrates by removing arabinose and acetyl side chains. Due to its specific characteristics, this enzyme seems to be of importance for industrial applications such as pretreatment of poultry cereals, bio-bleaching of wood pulp and degradation of plant biomass.

SGNH hydrolase-type esterase domain containing Cbes-AcXE2: a novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of many these enzymes is unknown. One of such enzymes is a predicted GDSL lipase or esterase encoded by the locus Athe_0553. In this study, this probable esterase named as Cbes-AcXE2 was overexpressed in Escherichia coli. The Ni-NTA affinity purified enzyme exhibited an optimum pH of 7.5 at an optimum temperature of 70 °C. Cbes-AcXE2 hydrolyzed p-nitrophenyl (pNP) acetate, pNP-butyrate, and phenyl acetate with approximately equal efficiency. The specific activity and K M for the most preferred substrate, phenyl acetate, were 142 U/mg and 0.85 mM, respectively. Cbes-AcXE2 removed the acetyl group of xylobiose hexaacetate and glucose pentaacetate like an acetyl xylan esterase (AcXE). Bioinformatics analyses suggested that Cbes-AcXE2, which carries an SGNH hydrolase-type esterase domain, is a member of an unclassified carbohydrate esterase (CE) family. Moreover, Cbes-AcXE2 is evolutionarily and biochemically similar to an unclassified AcXE, Axe2, of Geobacillus stearothermophilus. Thus, we proposed a novel family of carbohydrate esterase for both Cbes-AcXE2 and Axe2.

A novel bifunctional acetyl xylan esterase/arabinofuranosidase from Penicillium chrysogenum P33 enhances enzymatic hydrolysis of lignocellulose.

BACKGROUND: Xylan, the major constituent of hemicellulose, is composed of ß-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-L-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes. RESULTS: Here, we cloned an acetyl xylan esterase (PcAxe) from Penicillium chrysogenum P33 and expressed it in Pichia pastoris GS115. The optimal pH and temperature of the recombinant PcAxe (rPcAxe) for 4-nitrophenyl acetate were 7.0 and 40 °C, respectively. rPcAxe is stable across a broad pH range, retaining 100% enzyme activity om pH 6-9 after a 1 h incubation. The enzyme tolerates the presence of a wide range of metal ions. Sequence alignment revealed a GH62 domain exhibiting α-L-arabinofuranosidase activity with pH and temperature optima of pH 7.0 and 50 °C, in addition to the expected esterase domain. rPcAxe displayed significant synergy with a recombinant xylanase, with a degree of synergy of 1.35 for the hydrolysis of delignified corn stover. Release of glucose was increased by 51% from delignified corn stover when 2 mg of a commercial cellulase was replaced by an equivalent amount of rPcAxe, indicating superior hydrolytic efficiency. CONCLUSIONS: The novel bifunctional enzyme PcAxe was identified in P. chrysogenum P33. rPcAxe includes a carbohydrate esterase domain and a glycosyl hydrolase family 62 domain. This is the first detailed report on a novel bifunctional enzyme possessing acetyl xylan esterase and α-L-arabinofuranosidase activities. These findings expand our current knowledge of glycoside hydrolases and pave the way for the discovery of similar novel enzymes.

Replacement of carbohydrate binding modules improves acetyl xylan esterase activity and its synergistic hydrolysis of different substrates with xylanase.

BACKGROUND: Acetylation of the xylan backbone was a major obstacle to enzymatic decomposition. Removal of acetyl groups by acetyl xylan esterases (AXEs) is essential for completely enzymatic hydrolysis of xylan. Appended carbohydrate binding modules (CBMs) can promote the enzymatic deconstruction of plant cell walls by targeting and proximity effects. Fungal acetyl xylan esterases are strictly appended to cellulose-specific CBM1. It is still unclear whether xylan-specific CBMs have a greater advantage than CBM1 in potentiating the activity of fungal deacetylating enzymes and its synergistic hydrolysis of different substrates with xylanase. RESULTS: Three recombinant AXE1s fused with different xylan-specific CBMs, together with wild-type AXE1 with CBM1 and CBM1-deleted mutant AXE1dC, were constructed in this study. The optimal temperature and pH of recombinant AXE1s was 50 °C and 8.0 (except AXE1dC-CBM6), respectively. Cellulose-specific CBM1 in AXE1 obviously contributed to its catalytic action against substrates compared with AXE1dC. However, replacement of CBM1 with xylan-specific CBM4-2 significantly enhanced AXE1 thermostability and catalytic activity against soluble substrate 4-methylumbelliferyl acetate. Whereas replacements with xylan-specific CBM6 and CBM22-2 were more effective in enzymatic release of acetic acid from destarched wheat bran, NaClO2-treated wheat straw, and water-insoluble wheat arabinoxylan compared to AXE1. Moreover, replacement with CBM6 and CBM22-2 also resulted in higher degree releases of reducing sugar and acetic acid from different substrates when simultaneous hydrolysis with xylanase. A good linear relationship exists between the acetic acid and reducing sugar release. CONCLUSIONS: Our findings suggested that the replacement with CBM6 and CBM22-2 not only significantly improved the catalysis efficiency of AXE1, but also increased its synergistic hydrolysis of different substrates with xylanase, indicating the significance of targeting effect in AXE1 catalysis mediated by xylan-specific CBMs.

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a ß-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.

Regioselective deprotection of the monosaccharide-bearing thiocyanomethyl group at the anomeric position monitored by reversed-phase HPLC method.

In the current work, the investigation and development of a chemo-enzymatic approach for the synthesis of neo-glycoproteins have been studied. This strategy is based on the regioselective enzymatic hydrolysis of peracetylated monosaccharide, functionalized at the anomeric position (C1) as 1-thio-(S-cyanomethyl) group, a precursor of the 2- iminomethoxyethyl thioglycosides-linker for protein glycosylation, catalyzed by immobilized enzymes to obtain selectively monodeprotected compounds. The use of this activation in C1 is the most frequently used strategy for glycoprotein preparation. The selected biocatalysts are the lipase from Candida rugosa and the acetyl xylan esterase from Bacillus pumilus. A reversed-phase high-performance liquid-chromatographic (HPLC) method for monitoring the regioselective deprotection reaction has been developed. The developed HPLC method was used as a fingerprint to follow the hydrolysis of substrate 1 to substrate 1a and to determine its purity and yield. Moreover, the obtained compound was further purified by flash chromatography. The obtained compound 1a was further characterized using (1) H, (13) C NMR, correlation spectroscopy (COSY) and heteronuclear multiple bond correlation. The resulting product can be used as an intermediate for the preparation of di- and more complex oligosaccharides aimed at protein conjugation. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Heterologously Expressed Acetyl Xylan Esterase1 Isolated from the Anaerobic Rumen Fungus Neocallimastix frontalis PMA02.

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 ß-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

Xylanolytic enzyme systems in Arthrobacter sp. MTCC 5214 and Lactobacillus sp.

The production of extracellular xylanolytic enzymes such as xylanase, α-l-arabinofuranosidase (α-l-AFase), and acetyl xylan esterase (Axe) by marine Arthrobacter sp. and Lactobacillus sp. was investigated using different carbon sources. Induction and repression of all these enzymes differed with carbon source and also with the organism. Wheat bran was the best carbon source for the production of xylanase and α-l-AFase, whereas both isolates showed maximum Axe production when grown on oat bran as a carbon source. Preferential utilization of a carbon source for enzyme production can give us better insights into regulatory mechanism in these marine bacteria. Elution profile as well as zymogram analysis indicated the possibility of bifunctional α-l-AFase-Axes in both marine bacteria.

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

BACKGROUND: Lignin 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. RESULTS: Arabidopsis 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. CONCLUSIONS: Simultaneous 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.