Showing papers on "Esterase published in 2018"

Isolation of quercetin and mandelic acid from Aesculus indica fruit and their biological activities

Abstract: In this study Aesculus indica fruit was subjected to isolation of phytochemicals. Two antioxidants quercetin and Mandelic acid were isolated in pure state. The free radical scavenging and acetyl choline esterase inhibitory potential of the crude extract and sub fractions were also determined. The antioxidant capacity of crude extract, fractions and isolated compounds were determined by DPPH and ABTS methods. Folin-Ciocalteu reagent method was used to estimate the total phenolic contents and were found to be 78.34 ± 0.96, 44.16 ± 1.05, 65.45 ± 1.29, 37.85 ± 1.44 and 50.23 ± 2.431 (mg/g of gallic acid) in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fractions respectively. The flavonoid concentration in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fraction were; 85.30 ± 1.20, 53.80 ± 1.07, 77.50 ± 1.12, 26.30 ± 1.35 and 37.78 ± 1.25 (mg/g of quercetin) respectively. The chloroform fraction was more potent against enzymes, acetyl choline esterase and butyryl choline esterase (IC50 = 85 and 160 μg/ml respectively). The phenolic compounds in the crude extract and fractions were determined using HPLC standard method. Chlorogenic acid, quercetin, phloroglucinol, rutin, mandelic acid and hydroxy benzoic acid were detected at retention times 6.005, 10.062, 22.623, 30.597, 35.490 and 36.211 in crude extract and different fractions. The ethyl acetate fraction was rich in the targeted compounds and was therefore subjected to column isolation. The HPLC chromatogram of isolated compounds showed single peak at specified retention times which confirms their isolation in pure state. The isolated compounds were then characterized by FTIR and NMR spectrophotometric techniques. The Aesculus indica fruit extracts showed antioxidant and anticholine esterase inhibitory potentials. Two bioactive compounds were isolated in the pure form ethyl acetate fraction. From results it was concluded that the fruit of this plant could be used to minimize oxidative stress caused by reactive oxygen species.

Camel whey protein hydrolysates displayed enhanced cholesteryl esterase and lipase inhibitory, anti-hypertensive and anti-haemolytic properties

Abstract: Camel whey protein hydrolysates were generated using gastric (pepsin) and pancreatic (trypsin and chymotrypsin) enzymes for 3 and 6 h of hydrolysis time. Hydrolysates were characterized using degree of hydrolysis (DH) and reverse phase-high performance liquid chromatography (RP-HPLC). Camel whey protein hydrolysates (CWPHs) showed DH ranging from 11 to 47.5%, with chymotrypsin (6 h) and trypsin (3 h) generated hydrolysates exhibiting highest and lowest DH, respectively. HPLC analysis revealed that α-lactalbumin underwent complete degradation and newer shorter peptides were generated. Inhibition of cholesteryl esterase, (CE) pancreatic lipase and anti-hypertensive properties via angiotensin converting enzyme (ACE) inhibition were found to be higher in pepsin (6 h) and (3 h) generated CWPHs, respectively. Pepsin (P3 and P6) and trypsin (T6) generated hydrolysates exhibited highest anti-haemolytic activity followed by chymotrypsin generated CWPHs. Whereas, trypsin (T3) generated hydrolysate showed minimum anti-haemolytic activity (8.02% ± 1.40). Overall, CWPHs displayed enhanced inhibition of CE, lipase, ACE and anti-hemolytic properties upon hydrolysis, providing a strong scientific base for their potential to be considered as functional and nutraceutical ingredients. This study provides a strong indication of biologically active peptides responsible for health related bioactive properties reported herein. Future work must include identification of the peptides in the most potent camel whey protein hydrolysates.

New Diketopyrrolopyrrole-Based Ratiometric Fluorescent Probe for Intracellular Esterase Detection and Discrimination of Live and Dead Cells in Different Fluorescence Channels.

Abstract: A new diketopyrrolopyrrole-based fluorescent probe (DPP-AM) was designed and synthesized for ratiometric detection of esterase and for imaging of live and dead cells in different modes. DPP-AM showed red fluorescence because of the intramolecular charge transfer (ICT) process from the DPP moiety to the pyridinium cation and gave remarkable ratio changes (about 70 folds), with the fluorescence changing from red to yellow, after treating with esterase because of the broken ICT process. Besides, the detection limit of DPP-AM toward esterase in vitro was 9.51 × 10–5 U/mL. After pretreating with H2O2 and ultraviolet light radiation, the health status of TPC1 cells was successfully imaged. More importantly, DPP-AM showed yellow fluorescence in live cells and a red fluorescent signal in dead cells, indicating that DPP-AM has great potential applications for assessing esterase activity as well as for discriminating live and dead cells.

A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Abstract: Acetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive. An unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase. This study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.

The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch.

Abstract: Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin–carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. The data verify the enzyme’s unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism’s ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment.

Cloning, expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII

Abstract: A new esterase gene from thermophilic bacteria Ureibacillus thermosphaericus was cloned into the pET32b vector and expressed in Escherichia coli BL21(DE3). Alignment of the estUT1 amino acid sequence revealed the presence of a novel canonical pentapeptide (GVSLG) and 41–47% identity to the closest family of the bacterial lipases XIII. Thus the esterase estUT1 from U. thermosphaericus was assigned as a member of the novel family XVIII. It also showed a strong activity toward short-chain esters (C2–C8), with the highest activity for C2. When p-nitrophenyl butyrate is used as a substrate, the temperature and pH optimum of the enzyme were 70–80 °C and 8.0, respectively. EstUT1 showed high thermostability and 68.9 ± 2.5% residual activity after incubation at 70 °C for 6 h. Homology modeling of the enzyme structure showed the presence of a putative catalytic triad Ser93, Asp192, and His222. The activity of estUT1 was inhibited by PMSF, suggesting that the serine residue is involved in the catalytic activity of the enzyme. The purified enzyme exhibited high stability in organic solvents. EstUT1 retained 85.8 ± 2.4% residual activity in 30% methanol at 50 °C for 6 h. Stability at high temperature and tolerance to organic solvents make estUT1 a promising enzyme for biotechnology application.

Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S

Abstract: Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30–46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l−1 and 0.918 ± 0.025 U·µg−1, respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate-active enzymes (CAZy) database.

Abstract: Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell‐wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate‐active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes.

Amino-Acid-Based Metallo-Hydrogel That Acts Like an Esterase

Abstract: A histidine-based amphiphile containing a C14 fatty acyl chain, N- histidyl N′-myristry ethyl amine (AM1, 14.7 mM) forms hydrogels in the presence of Fe3+ (within the range 1.47 to 4.41 mM) and Hg2...

A dual fluorogenic and 19F NMR probe for the detection of esterase activity

Abstract: In this study, a dual-channel probe of FlE based on flavonoid derivatives is reported, which can yield “turn-on” signals of both fluorometry and 19F nuclear magnetic resonance in response to the presence of esterase. Upon the addition of esterase, FlE could convert into Fl, possessing the ESIPT effect. As a result, the fluorescence intensity was significantly enhanced and peaked at 510 nm, accompanying a change in the fluorescence of solution from nonluminous to strong green. Moreover, as determined by 19F NMR, the signal could apparently shift from δF −111.57 to −111.69 ppm. Due to the combination of these two detection approaches having good sensitivity, high selectivity, and real-time detection, FlE has been successfully applied to confocal fluorescence imaging for the detection of esterase in cells, which consistent with the 19F NMR test results in some sense.

Preparation of Stable Cross-Linked Enzyme Aggregates (CLEAs) of a Ureibacillus thermosphaericus Esterase for Application in Malathion Removal from Wastewater

Abstract: In this study, the active and stable cross-linked enzyme aggregates (CLEAs) of the thermostable esterase estUT1 of the bacterium Ureibacillus thermosphaericus were prepared for application in malathion removal from municipal wastewater. Co-expression of esterase with an E. coli chaperone team (KJE, ClpB, and ELS) increased the activity of the soluble enzyme fraction up to 200.7 ± 15.5 U mg−1. Response surface methodology (RSM) was used to optimize the preparation of the CLEA-estUT1 biocatalyst to maximize its activity and minimize enzyme loss. CLEA-estUT1 with the highest activity of 29.4 ± 0.5 U mg−1 (90.6 ± 2.7% of the recovered activity) was prepared with 65.1% (w/v) ammonium sulfate, 120.6 mM glutaraldehyde, and 0.2 mM bovine serum albumin at 5.1 h of cross-linking. The biocatalyst has maximal activity at 80 °С and pH 8.0. Analysis of the properties of CLEA-estUT1 and free enzyme at 50–80 °C and pH 5.0–10.0 showed higher stability of the biocatalyst. CLEA-estUT1 showed marked tolerance against a number of chemicals and high operational stability and activity in the reaction of malathion hydrolysis in wastewater (up to 99.5 ± 1.4%). After 25 cycles of malathion hydrolysis at 37 °С, it retained 55.2 ± 1.1% of the initial activity. The high stability and reusability of CLEA-estUT1 make it applicable for the degradation of insecticides.

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica

Abstract: Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s−1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.

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

Abstract: 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 A, 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/β hydrolase enzymes.

Synergistic effect of mutagenesis and truncation to improve a polyesterase from Clostridium botulinum for polyester hydrolysis.

Abstract: The activity of the esterase (Cbotu_EstA) from Clostridium botulinum on the polyester poly(ethylene terephthalate) (PET) was improved by concomitant engineering of two different domains. On the one hand, the zinc-binding domain present in Cbotu_EstA was subjected to site-directed mutagenesis. On the other hand, a specific domain consisting of 71 amino acids at the N-terminus of the enzyme was deleted. Interestingly, a combination of substitution of residues present in the zinc-binding domain (e.g. S199A) synergistically increased the activity of the enzyme on PET seven fold when combined to the truncation of 71 amino acids at the N-terminus of the enzyme only. Overall, when compared to the native enzyme, the combination of truncation and substitutions in the zinc-binding domain lead to a 50-fold activity improvement. Moreover, analysis of the kinetic parameters of the Cbotu_EstA variants indicated a clear shift of activity from water soluble (i.e. para-nitrophenyl butyrate) to insoluble polymeric substrates. These results evidently show that the interaction with non-natural polymeric substrates provides targets for enzyme engineering.

The application of digestive tract lactic acid bacteria with high esterase activity for zearalenone detoxification.

Abstract: Zearalenone (ZEA) is an estrogenic mycotoxin produced by several Fusarium species and frequently contaminates cereals used for food or animal feed. This study attempted to select lactic acid bacteria (LAB) with high esterase activity from the digestive tract, with the goal of using these bacteria for ZEA detoxification.; Results: No ZEA activity-related biotransformation products were observed in three isolates (B1, B2 and D10) during incubation in the presence of ZEA. All three LAB strains were Lactobacillus plantarum, but the API 50 CHL results suggested that the three isolates were different strains. Increased esterase activity was associated with an increase in cell growth, and the ZEA-detoxifying capabilities of isolates rely on the concentration of bacteria in the culture medium. The lipolytic activity and ZEA removal assay indicated that ZEA degradation by the supernatant fraction was dependent on esterase activity; the supernatant of B2 strain showed the highest ZEA degradation ability and did not release the binding ZEA back into the medium. The D10 strain showed fast ZEA binding ability during the late log phase but began to release the bound ZEA back into the medium after the early stationary phase. All isolates showed good acid and bile salt tolerance ability but all strains showed low adhesion ability to epithelial cells.; Conclusion: Based on the ZEA removal characterization and ability of the isolates, it is suggested that the isolates could be applied to ZEA detoxification of contaminated feed, but the with the requirement of high cell number for ZEA binding and limited degradation time before absorption of ZEA in the digestive tract. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

Characterisation of a Hydroxycinnamic Acid Esterase From the Bifidobacterium longum subsp. longum Taxon

Abstract: Bifidobacterium longum subsp. longum, a common member of the human gut microbiota with perceived positive health effects, is capable of metabolising certain complex, plant-derived carbohydrates which are commonly found in the (adult) human diet. These plant glycans may be employed to favourably modulate the microbial communities in the intestine. Hydroxycinnamic acids (HCAs) are plant phenolic compounds, which are attached to glycans, and which are associated with anti-oxidant and other beneficial properties. However, very little information is available regarding metabolism of HCA-containing glycans by bifidobacteria. In the current study, a gene encoding a hydroxycinnamic acid esterase was found to be conserved across the B. longum subsp. longum taxon and was present in a conserved locus associated with plant carbohydrate utilisation. The esterase was shown to be active against various HCA-containing substrates and was biochemically characterised in terms of substrate preference, and pH and temperature optima of the enzyme. This novel hydroxycinnamic acid esterase is presumed to be responsible for the release of HCAs from plant-based dietary sources, a process that may have benefits for the gut environment and thus host health.

Characterization and inhibition effects of some metal ions on carbonic anhydrase enzyme from Kangal Akkaraman sheep

Abstract: In this work, the carbonic anhydrase (CA) enzyme was purified from Kangal Akkaraman sheep in Sivas, Turkey with specific activity value of 6681.57 EU/mg and yield of 14.90% with using affinity column chromatography. For designating the subunit molecular mass and enzyme purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis method was used and single band for this procedure was obtained. The molecular mass of CA enzyme was found as 28.89 kDa. In this study, the optimum temperature and optimum pH were obtained from 30 and 7.5. Vmax and Km values for p-nitrophenylacetate substrate of the CA were determined from Lineweaver-Burk graphs. Additionally, the inhibitory results of diverse heavy metal ions (Hg+ , Fe2+ , Pb2+ , Co2+ , Ag+ , and Cu2+ ) on sheep were studied. Indeed, CA enzyme activities of Kangal sheep were investigated with using esterase procedure under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC50 ) and Ki values were obtained.

A novel glucuronoyl esterase from Aspergillus fumigatus-the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters.

Abstract: Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of d-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40–50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.

Action of different types of endoxylanases on eucalyptus xylan in situ

Abstract: Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.