Optimization of reaction conditions for enzymatic viscosity reduction and hydrolysis of wheat arabinoxylan in an industrial ethanol fermentation residue

This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 degrees C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76-84%, 75-80%, and 43-47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, beta-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2-6.4 and 41-49 degrees C for arabinose release and from pH 4.9-5.3 and 42-46 degrees C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields.     

Enzymatic hydrolysis of water-soluble wheat arabinoxylan. 1. Synergy between alpha-L-arabinofuranosidases,endo-1,4-beta-xylanases,and beta-xylosidase activities

Hydrolysis of arabinoxylan is an important prerequisite for improved utilization of wheat hemicellulose in the ethanol fermentation industry. This study investigates the individual and combined efficiencies of three commercial, cellulytic and hemicellulytic enzyme preparations, Celluclast 1.5 L, Ultraflo L, and Viscozyme L, in catalyzing the liberation of arabinose and xylose from water-soluble wheat arabinoxylan. Ultraflo L was the best enzyme preparation for releasing arabinose, liberating 53 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 40 degrees C, pH 6). Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose, liberating 26 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 50 degrees C, pH 5). The 50:50 mixtures of the enzyme preparations showed no synergistic cooperation in arabinose release, but a synergistic interaction in xylose release was found between Ultraflo L and Celluclast 1.5 L. On the basis of high-performance anion exchange chromatography (HPAEC) analysis of the hydrolysates after enzymatic reaction, we propose that the observed synergism between Celluclast 1.5 L and Ultraflo L is the result of positive interaction between alpha-L-arabinofuranosidase and endo-1,4-beta-xylanase activities present in Ultraflo L that released arabinose, xylobiose and xylotriose, and beta-xylosidase activities in Celluclast 1.5 L, capable of catalyzing the hydrolysis of xylobiose and xylotriose to xylose.     

Identification and characterization of a novel arabinoxylanase from wheat flour.

An endogenous wheat (Triticum aestivum) flour endoxylanase was purified to homogeneity from a crude wheat flour extract by ammonium sulfate precipitation and cation-exchange chromatography. The 30-kD protein had an isoelectric point of 9.3 or higher. A sequence of 19 amino acids at the NH2 terminus showed 84.2% identity with an internal sequence of 15-kD grain-softness protein, friabilin. High-performance anion-exchange chromatography and gel-permeation analysis of the hydrolysis products indicated the preferential hydrolysis of highly branched structures by the enzyme; wheat arabinoxylan and rye (Secale cereale) arabinoxylan (high arabinose to xylose ratios) were hydrolyzed more efficiently by this enzyme than oat (Avena sativa) spelt xylan (low arabinose to xylose ratios). The release of the hydrolysis products as a function of time suggested that the endoxylanolytic activity was associated with the release of arabinose units from the polysaccharides, suggesting that the enzyme action is similar to that by endoxylanases from Ceratocystis paradoxa, Aspergillus niger, and Neurospora crassa. Although the enzyme released arabinose from arabinoxylan, it did not hydrolyze p-nitrophenyl-alpha-L-arabinofuranoside. From the above, it follows that the enzyme, called arabinoxylanase, differs from most microbial endoxylanases and from an endoxylanase purified earlier from wheat flour.     

Distinct Actions by Paenibacillus sp. Strain E18 α-l-Arabinofuranosidases and Xylanase in Xylan Degradation

We cloned a Paenibacillus sp. strain E18 5.3-kb xylanolytic gene cluster that contains three open reading frames encoding two family 43 α-l-arabinofuranosidases (Abf43A and Abf43B) and one family 10 xylanase (XynBE18). The deduced amino acid sequences of Abf43A and Abf43B were at most 68% and 63% identical to those of two putative family 43 proteins from Clostridium sp. strain DL-VIII ({"type":"entrez-protein","attrs":{"text":"EHI98634.1","term_id":"357170460","term_text":"EHI98634.1"}}EHI98634.1 and {"type":"entrez-protein","attrs":{"text":"EHI98635.1","term_id":"357170461","term_text":"EHI98635.1"}}EHI98635.1), respectively, but were only 11% identical to each other. Recombinant Abf43A and Abf43B had similar activities at 45°C and pH 6.0 but varied in thermostabilities and substrate specificities. Abf43B was active against only 4-nitrophenyl α-l-arabinofuranoside, whereas Abf43A acted on 4-nitrophenyl α-l-arabinofuranoside, wheat arabinoxylan, 4-nitrophenyl α-d-xylopyranoside, and sugar beet arabinan. The sequential and combined effects on xylan degradation by XynBE18, Abf43A, and Abf43B were characterized. For beechwood, birchwood, and oat spelt xylans as the substrates, synergistic effects were found when XynBE18 and Abf43A or Abf43B were incubated together and when the substrates were first incubated with Abf43A or Abf43B and then with XynBE18. Further high-performance liquid chromatography (HPLC) analysis showed that the amounts of xylobiose and xylose increased sharply in the aforementioned reactions. For water-soluble wheat arabinoxylan as the substrate, Abf43A not only released arabinose but also had a synergistic effect with XynBE18. Synergy may arise as the result of removal of arabinose residues from xylans by α-l-arabinofuranosidases, which eliminates steric hindrance caused by the arabinose side chains and which allows xylanases to then degrade the xylan backbone, producing short xylooligosaccharides.     

Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan

AIMS: To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. METHODS AND RESULTS: The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. CONCLUSIONS: The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.     

Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei), Finizym (from Aspergillus niger), Ultraflo L (from Humicola insolens), and Viscozyme L (from Aspergillus aculeatus) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60 °C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60 °C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50 °C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α-l-arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations.     

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

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

A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley analysis of substrate preference and specificity by nano-probe NMR.

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of approximately 60 and approximately 66 kDa, a pI of 4.55 and almost identical N-terminal amino-acid sequences. With p-nitrophenyl alpha-L-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a Km of 0.5 mM and a Vmax of 6.7 micromol. min-1.(mg of protein)-1. Maximum activity was displayed at pH 4.2 and 60 degrees C, and, under these conditions, the half-life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p-nitrophenyl alpha-L-arabinopyranoside or p-nitrophenyl beta-D-xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released arabinose from high-molecular-mass arabinoxylan and arabinoxylan-derived oligosaccharides but was inactive against linear or branched-chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo-oligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substituents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-O-linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non-reducing terminal xylose, the 3-O-linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p-nitrophenyl alpha-L-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.     

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.     

Structural studies on water-soluble arabinoxylans in rye grain using enzymatic hydrolysis

The major water-soluble arabinoxylan fraction from rye grain, containing 4-linked β- -xylopyranosyl residues of which about 43% were substituted solely at O-3 and 7% at both O-2 and O-3 with terminal - -arabinofuranosyl units, was hydrolysed to different extents using semi-purified xylanase from Trichoderma reesei. Products were fractionated on Biogel P-2 and structurally elucidated by sugar, methylation and high-field ¹H-NMR analysis. Moderate hydrolysis released arabinose, xylose, xylobiose, xylotriose and xylotetraose together with xylo-oligosaccharides (DP ≥ 4) in which one or more of the residues were substituted at O-3 with a terminal arabinose unit. The xylose residues substituted with arabinose units at both O-2 and O-3 became enriched in the remaining polymeric fraction. Extensive hydrolysis with the enzyme released arabinose, xylose and xylobiose as major products together with small amounts of two oligosaccharides and a polymeric fraction. One of the oligosaccharides was identified as xylotriose in which the non-reducing end was substituted at O-2 and O-3 with terminal arabinose units and the other as xylotetraose in which one of the interjacent residues was substituted with arabinose units in the same way. The polymeric fraction contained a main chain of 4-linked xylose residues in which 60–70% of the residues were substituted at both O-2 and O-3 with arabinose units.

The semi-purified enzyme contained xylanase and arabinosidase activities which rapidly degraded un- and mono-substituted xylose residues while the degradation of double-substituted xylose residues was much slower. The results show that the mono- and double-substituted xylose residues were present in different polymers or different regions of the same polymer.     

Studies of the polysaccharides from sunflower heads

Extraction of sunflower heads with ammonium oxalate afforded water-soluble pectin material and water-insoluble glycoprotein material, the carbohydrate portion of which consisted of galacturonic acid and xylose residues; the pectin material defied fractionation with cetylpyridinium chloride. Extraction with hydrochloric acid (pH 1.5) afforded water-soluble and water-insoluble polysaccharide materials. The former, when fractionated with cetylpyridinium chloride, gave a glycoprotein, the carbohydrate moiety of which was composed of galacturonic acid, galactose (major), glucose, arabinose, and xylose, and also a rhamnan. The latter was a glycoprotein, the carbohydrate portion of which consisted of galactose (major), glucose, xylose, and rhamnose residues. Extraction of the sunflower heads with water also gave glycoprotein material, which was fractionated by paper electrophoresis into a glyco-protein, the carbohydrate moiety ofwhich was composed of galacturonic acid (minor), galactose, glucose, xylose, arabinose, and rhamnose (major) residues, and a heteropolysaccharide composed of galactose (major), glucose, xylose, and arabinose residues.     

Endogeneous β-<Emphasis Type="SmallCaps">d</Emphasis>-xylosidase and α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase activity in flax seed mucilage

Flax seed mucilage (FM) contains a mixture of highly doubly substituted arabinoxylan as well as rhamnogalacturonan I with unusual side group substitutions. Treatment of FM with a GH11 Bacillus subtilis XynA endo 1,4-β-xylanase (BsX) gave limited formation of reducing ends but when BsX and FM were incubated together on different wheat arabinoxylan substrates and birchwood xylan, significant amounts of xylose were released. Moreover, arabinose was released from both water-extractable and water-unextractable wheat arabinoxylan. Since no xylose or arabinose was released by BsX addition alone on these substrates, nor without FM or BsX addition, the results indicate the presence of endogenous β-d-xylosidase and α-l-arabinofuranosidase activities in FM. FM also exhibited activity on both p-nitrophenyl α-l-arabinofuranoside (pNPA) and p-nitrophenyl β-d-xylopyranoside (pNPX). Based on K _M values, the FM enzyme activities had a higher affinity for pNPX (K _M 2 mM) than for pNPA (K _M 20 mM).     

Feruloyl oligosaccharides stimulate the growth of Bifidobacterium bifidum

Insoluble dietary fiber from wheat bran contains some feruloyl groups linked to the arabinose residues in the cell wall arabinoxylan. Treatment of wheat bran insoluble dietary fiber with xylanase from Bacillus subtilis yielded feruloyl oligosacchairdes, which were purified with Amberlite XAD-2. Saponification of the feruloyl oligosaccharides released ferulic acid and arabinoxylan oligosaccharides which consist of arabinose and xylose. The effect of the feruloyl oligosacchairdes on the growth of Bifidobacterium bifidum F-35 was investigated in vitro. The B. bifidum produced acid when cultivated anaerobically in TPY broth with 0.5% feruloyl oligosacchairdes as the carbohydrate source. The biomass yield of the B. bifidum increased with increasing the concentration of feruloyl oligosaccharides in TPY broth. The maximum cell growth was increased by 50% in TPY broth supplemented with 0.1% feruloyl oligosaccharides compared to TPY broth. These results indicated that the growth of B. bifidum F-35 was promoted by the feruloyl oligosaccharides from wheat bran insoluble dietary fiber, and not suppressed by the ferulic acid moiety of them.     

Chrysosporium lucknowense C1 arabinofuranosidases are selective in releasing arabinose from either single or double substituted xylose residues in arabinoxylans

Two novel arabinofuranosidases, Abn7 and Abf3 from Chrysosporium lucknowense (C1), belonging to the glycoside hydrolase family 43 and 51 were purified and characterized. Abn7 is exclusively able to hydrolyze arabinofuranosyl residues at position O-3 of double substituted xylosyl residues in arabinoxylan-derived oligosaccharides, an activity rarely found thus far. Abf3 is able to release arabinose from position O-2 or O-3 of single substituted xyloses. Both enzymes performed optimal at pH 5.0 and 40°C. Combining Abn7 and Abf3 resulted in a synergistic increase in arabinose release from arabinoxylans. This synergistic effect is due to the action of Abf3 on the remaining arabinose residues at position O-2 on single substituted xylosyl residues resulting from the action of Abn7 on double substituted xylosyl residues. Arabinose release was further increased when an endo-1,4-β-xylanase was present during digestion. The efficiency of these arabinohydrolases from C1 on insoluble arabinoxylan substrates is discussed.     

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp.

A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. © Rapid Science Ltd. 1998     

Periodate oxidation and degradation studies on the major water-soluble arabinoxylan in rye grain

The major water-soluble arabinoxylan from rye grain has previously been shown to contain a main chain of 4-linked β- -xylopyranosyl residues in which, on average, every second is substituted at position 3 with terminal - -arabinofuranosyl residues. Periodate oxidation, reduction and fragmentation by mild acid hydrolysis produced a series of glycerol xylosides containing 4-linked xylopyranosyl residues linked at the reducing end to position 2 of glycerol. It was shown that a one-step periodate oxidation was incomplete due to the formation of relatively stable hemiacetal linkages. A sequential oxidation and reduction procedure was used to bring about complete oxidation of arabinose and unbranched xylose residues in the intact polysaccharide. Quantitative analysis of the products liberated by mild acid hydrolysis revealed the presence of glycerol xylosides with one, two or three xylose residues in the molar ratio of 1·00:0·86:0·02. The xylose residues must have originated from branched residues in the main chain of the arabinoxylan. The units or small blocks of two residues are therefore distributed mainly as isolated branched residues and not randomly as previously reported.     

Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

Endoxylanase substrate selectivity determines degradation of wheat water-extractable and water-unextractable arabinoxylan

The relative activity of an endoxylanase towards water-unextractable (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, referred to as endoxylanase substrate selectivity, impacts the enzyme functionality in cereal-based biotechnological processes such as bread-making and gluten starch separation. A set of six endoxylanases representing a range of substrate selectivities as determined by a screening method using chromophoric substrates [Anal. Biochem.2003, 319, 73-77] was used to examine the impact of such selectivity on changes in structural characteristics of wheat WU-AX and WE-AX upon enzymic hydrolysis. While WE-AX degradation by the selected endoxylanases was very comparable with respect to apparent molecular mass (MM) profiles and arabinose to xylose ratio of the hydrolysates formed, WU-AX solubilisation and subsequent degradation of solubilised fragments gave rise to widely varying MM profiles, depending on the substrate selectivity of the enzymes. Enzymes with high selectivity towards WU-AX de facto generated higher MM fragments from WU-AX than enzymes with low selectivity. The arabinose to xylose ratios of solubilised fragments were independent of the degree of solubilisation.     

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

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

pH catalyzed pretreatment of corn bran for enhanced enzymatic arabinoxylan degradation

Corn bran is mainly made up of the pericarp of corn kernels and is a byproduct stream resulting from the wet milling step in corn starch processing. Through statistic modeling this study examined the optimization of pretreatment of corn bran for enzymatic hydrolysis. A low pH pretreatment (pH 2, 150 °C, 65 min) boosted the enzymatic release of xylose and glucose and maximized biomass solubilization. With more acidic pretreatment followed by enzymatic hydrolysis the total xylose release was maximized (at pH 1.3) reaching ～ 50% by weight of the original amount present in destarched corn bran, but the enzyme catalyzed xylose release was maximal after pretreatment at approx. pH 2. The total glucose release peaked after pretreatment of approx. pH 1.5 with an enzymatic release of approx. 68% by weight of the original amounts present in destarched corn bran. For arabinose the enzymatic release was negatively affected by the acidic pretreatment as labile arabinosyl-linkages were presumably hydrolysed directly during the pretreatment. A maximum of 60% arabinose release was achieved directly from the optimal (acidic) pretreatment. The total content of diferulic acids, supposedly involved in the cross-linking of the arabinoxylan polymers, decreased by both alkaline and acidic pretreatment pH, with the loss by alkaline pretreatments being highest. No direct correlation between the enzymatic release of xylose and the content of diferulic acids in the substrate could be verified. On the contrary the enzymatic release of xylose was significantly correlated to the total release of arabinose, indicating that the degree of arabinosyl-substitutions on the xylan backbone is an essential parameter for enzymatic hydrolysis of corn bran arabinoxylan.