Hydrolysis Of Xylooligosaccharides Research Articles

Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus (Hungateiclostridium, Clostridium clariflavum). Clocl_1795 has been shown to be a xylobiohydrolase AcXbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated AcXyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that AcXyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. AcXyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that AcXyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. KEY POINTS: Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily.

Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

Crystal structure and identification of amino acid residues for catalysis and binding of GH3 AnBX β-xylosidase from Aspergillus niger

β-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 β-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (β/α)8 TIM-barrel-like domain, an (α/β)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. KEY POINTS: • Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively • Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX • The W86A and W86S mutations in AnBX increased the preference for glucoside substrates.

Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity.

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear β-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA2Xyln. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.

Contributions and characteristics of two bifunctional GH43 β-xylosidase /α-L-arabinofuranosidases with different structures on the xylan degradation of Paenibacillus physcomitrellae strain XB

Xylan is one of the major polymeric hemicellulosic constituents of lignocellulosic biomass, and its effective utilization by microorganisms is crucial for the economical production of biofuels. In this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability on different substrates of corncob xylan (CCX), oat spelt xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). The RT-QPCR result showed that two genes (Pph_0602 and Pph_2344) belonging to the glycoside hydrolase family 43 were up-regulated more than 5-fold on CCX and xylose. Substrate-specific assays with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both exhibited β-xylosidase activity toward the chromogenic substrate p-nitrophenyl-β-D-xylopyranoside, and α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside, indicating their bifunctionality. By testing their degradation characteristics on different natural substrates, it was found that both Ppxyl43A and Ppxyl43B showed similar degradation ability on CCX and OSX. Both enzymes could hydrolyze xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX, suggesting they mainly hydrolyze xylo-oligosaccharides by β-xylosidase activity. Further analysis showed that both of them displayed very high pH stability and thermostability on the β-xylosidase activity, but Ppxy143B exhibited wider pH and temperature ranges, higher pH and temperature stability, was less influenced by metal ions, and had a slower start-up response than Ppxyl43A. Given their predicted structure, it is likely that the enzymatic differences between Ppxyl43A and Ppxyl43B might be related to the extra C-terminus domain (GH43_C2) in Ppxyl43B, which could enhance the enzymatic stability while restricting the substrates’ or metal ions’ access to the active sites of Ppxyl43B. In conclusion, both Ppxyl43A and Ppxyl43B were β-xylosidase/α-L-arabinofuranosidase bifunctional enzymes and might be useful in xylan biomass conversion, especially in the hydrolysis of xylo-oligosaccharides into xylose.

Degradation Techniques of Hemicellulose Fraction from Biomass Feedstock for Optimum Xylose Production: A Review

The utilization of xylose, one of the C-5 sugars released from hemicellulose deconstruction of biomass feedstock, is intensively studied, in particular for producing valuable fermentation products. More xylose in the initial fermentation broth is highly expected to induce more final products formed. The techniques for lignocellulose fractionation to obtain the expected xylose amount are widely developed. Nevertheless, these are slightly different from the methods for glucose production. The production of glucose is initiated by delignification to facilitate enzyme accessibility in degrading cellulose to glucose. The delignification process, however, requires harsh conditions causing further decomposition of xylose corresponded to the formation of inhibitors detected in the spent liquor of pretreatment. Hence, xylose recovery needs particular treatments and the corresponding condition. Several strategies offered attributed to xylose production involved in conventional treatment using dilute acid and other promising methods such as an autohydrolysis, solid acids, steam explosion, inorganic salt, and ionic liquid. Post-treatments by enzymatic or acid hydrolysis of xylooligosaccharides dissolved in the liquid fraction are also considered to enhance xylose recovery. This review provides such techniques and the challenges frequently arising during xylose yield optimization performed. Each method has specific benefits and drawbacks that are also addressed in this paper.

Taxogenomic assessment and genomic characterisation of Weissella cibaria strain 92 able to metabolise oligosaccharides derived from dietary fibres

The importance of the gut microbiota in human health has led to an increased interest to study probiotic bacteria. Fermented food is a source of already established probiotics, but it also offers an opportunity to discover new taxa. Four strains of Weissella sp. isolated from Indian fermented food have been genome sequenced and classified into the species W. cibaria based on whole-genome phylogeny. The genome of W. cibaria strain 92, known to utilise xylooligosaccharides and produce lactate and acetate, was analysed to identify genes for oligosaccharide utilisation. Clusters including genes involved in transportation, hydrolysis and metabolism of xylooligosaccharides, arabinooligosaccharides and β-glucosides were identified. Growth on arabinobiose and laminaribiose was detected. A 6-phospho-β-glucosidase clustered with a phosphotransferase system was found upregulated during growth on laminaribiose, indicating a mechanism for laminaribiose utilisation. The genome of W. cibaria strain 92 harbours genes for utilising the phosphoketolase pathway for the production of both acetate and lactate from pentose and hexose sugars but lacks two genes necessary for utilising the pentose phosphate pathway. The ability of W. cibaria strain 92 to utilise several types of oligosaccharides derived from dietary fibres, and produce lactate and acetate makes it interesting as a probiotic candidate for further evaluation.

Biohydrogen production using xylose or xylooligosaccharides derived from sugarcane bagasse obtained by hydrothermal and acid pretreatments

The employment of hemicellulose fractions from bagasse acid (HF-AC - rich in xylose) and hydrothermal (HF-HT - containing high amounts of xylooligosaccharides) pretreatments led to yields of 265 and 177 mLH2/gcarbohydrate after 18 and 45 h of fermentation, respectively. The anaerobic sludge used as inoculum was able to hydrolyze the xylooligosaccharides of HF-HT into simpler sugars, enabling microbial hydrogen production. The acid hydrolysis of xylooligosaccharides in HF-HT before fermentation improved the productivity by 30% and showed similar yield (231 mLH2/gcarbohydrate). The adaptive phase for HF-HT consumption was influenced by the hydrolytic step and by inhibitors, as the kinetics of hydrolyzed HF-HT was deeply affected in the first 18 h compared to HF-AC. Higher amounts of HF-AC in the media promoted fermentation inhibition, which could be related to the increased concentrations of acetic acid and carbohydrates, two factors that enhance the system osmolarity.

GH11 xylanase from Aspergillus tamarii Kita: Purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry

The present study describes the one-step purification and biochemical characterization of an endo-1,4-β-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0–9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20μmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds β-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.

β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization, and application for xylooligosaccharide hydrolysis

The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50 °C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55 °C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were Vmax 0.20 μmol.min−1 mg prot.−1, Km 0.45 mM, Kcat 0.82 s−1, and Kcat/Km 1.82 s−1 mM−1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.

THE PRELIMINARY STUDY OF ANTIOXIDANT ACTIVITY FROM XYLO-OLIGOSACCHARIDE OF CORNCOB (ZEA MAYS) HYDROLYSIS PRODUCT WITH ENDO-β-XYLANASE ENZYME

Xylo-oligosaccharide derived from corncob hemicellulose has been reported to possess antioxidant activity. In order to assess the effective scavenging of xylo-oligosaccharide, we conducted in vitro studies based on self-made xylo-oligosaccharide with DPPH (2,2diphenyl-1-picrilhydrazil) method. Xylo-oligosaccharide was prepared with enzymatic hydrolysis. The enzyme used for hemicellulose hydrolysis was endo-β-xylanase enzyme from PC-01 isolated bactrerium. PC-01 isolated bacterium used in this study was Pacet hot spring which was isolated from East Java. Endo-β-xylanase enzyme is an extracelluler enzyme. There was about 0.199 U/mL after purification and dialysis process. Hydrolisis product of hemicellulose A and B from corncob were analyzed with TLC (Thin Layer Chromatography) and HPLC (High Performance Liquid Chromatography). This analysis showed that hydrolysis product of hemicellulose B had a lot of xylo-oligosaccharide hydrolysis product of hemicellulose than Xylo-oligosaccharide hydrolysis product of hemicelluloses A. Xylo-olygosaccharide was analyzed as on antioxidant activity. Xylo-oligosaccharide hydrolysis product ofhemicellulose B (IC = 48.96) has higher antioxidant activity than Xylo-oligosaccharide hydrolysis product of hemicellulose A (IC 50 50 = 92.302). The toxicity of xylo-oligosaccharide can be calculated by the value of LC 50 (Lethality concentration). LC of xylooligosaccharide derived from corncob hemicellulose was 400 ppm so that xylo-oligosaccharide has anti tumor activity because xylooligosaccharide has LC 50 < 1000 ppm.

Hydrolysis kinetics of tulip tree xylan in hot compressed water

Lignocellulosic biomass, a promising renewable resource, can be converted into numerous valuable chemicals post enzymatic saccharification. However, the efficacy of enzymatic saccharification of lignocellulosic biomass is low; therefore, pretreatment is necessary to improve the efficiency. Here, a kinetic analysis was carried out on xylan hydrolysis, after hot compressed water pretreatment of the lignocellulosic biomass conducted at 180–220°C for 5–30min, and on subsequent xylooligosaccharide hydrolysis. The weight ratio of fast-reacting xylan to slow-reacting xylan was 5.25 in tulip tree. Our kinetic results were applied to three different reaction systems to improve the pretreatment efficiency. We found that semi-continuous reactor is promising. Lower reaction temperatures and shorter space times in semi-continuous reactor are recommended for improving xylan conversion and xylooligosaccharide yield. In the theoretical calculation, 95% of xylooligosaccharide yield and xylan conversion were achieved simultaneously with high selectivity (desired product/undesired product) of 100 or more.

Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state.

Trichoderma reesei (Tr.) cellulases, which convert cellulose to reducing sugars, are a promising catalyst used in the lignocellulosic biofuel production. Improving Tr. cellulases activity, though very difficult, is highly desired due to the recalcitrance of lignocellulose. Meanwhile, it is preferable to enhance the cellulase's promiscuity so that substrates other than cellulose can also be hydrolyzed. In this work, an attempt is made to improve the catalytic activity of a major endogluanase Tr. Cel7B against xylan which crosslinks with cellulose in lignocellulose. By using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the transition state of the xylo-oligosaccharide hydrolysis is identified. Then, mutations are introduced and their effect on the transition state stabilization is ranked based on the free energy calculations. Seven top ranked mutants are evaluated experimentally. Three mutants A208Q, A222D, and G230R show a higher activity than the wild-type Tr. Cel7B in the hydrolysis of xylan (by up to 47%) as well as filter paper (by up to 50%). The combination of the single mutants can further improve the enzyme activity. Our work demonstrates that the free energy method is effective in engineering the Tr. Cel7B activity against xylan and cellulose, and thus may also be useful for improving the activity of other Tr. cellulases. Biotechnol. Bioeng. 2016;113: 1171-1177. © 2015 Wiley Periodicals, Inc.

Molecular cloning, overexpression, purification and crystallographic analysis of a GH43 β-xylosidase from Bacillus licheniformis.

β-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family.

Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu(2+) tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and Km and Vmax values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.

A novel β-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds.

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of the G. thermoglucosidasius β-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

Four GH11 xylanases from the xylanolytic fungus Talaromyces versatilis act differently on (arabino)xylans

The filamentous fungus Talaromyces versatilis produces a wide range of cellulolytic and hemicellulolytic enzymes such as xylanases. The recent accessibility to the T. versatilis genome allows identifying two new genes, xynE and xynF, encoding glycoside-hydrolases from family GH11. Both genes were cloned and expressed in the methylotrophic yeast Pichia pastoris in order to compare these new xylanases with two other GH11 xylanases from T. versatilis (XynB and XynC) that were previously reported. High-level expression of recombinant enzymes was obtained for the four enzymes that were purified to homogeneity. The XynB, XynC, XynE and XynF enzymes have molecular masses of 34, 22, 45 and 23 kDa, an optimal pH between 3.5 and 4.5 and an optimal temperature between 50 °C and 60 °C. Interestingly, XynF has shown the best thermal stability at 50 °C for at least 180 min with a weak loss of activity. The four xylanases catalysed hydrolysis of low viscosity arabinoxylan (LVAX) with K m(app) between 11.5 and 23.0 mg.mL(-1) and k cat/K m(app) 170 and 3,963 s(-1) mg(-1).mL. Further investigations on the rate and pattern of hydrolysis of the four enzymes on LVAX showed the predominant production of xylose, xylobiose and some (arabino)xylo-oligosaccharides as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing degree of polymerisation of oligomer up to 6, suggesting that the specificity region of XynE and XynF spans at least six xylose residues. Because of their attractive properties, T. versatilis xylanases might be considered for biotechnological applications.

A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

Paenibacillus woosongensis의 Xylanase 유전자 클로닝과 특성분석

Paenibacillus woosongensis의 유전체 부분 염기서열로부터 유추된 xylanase 유전자를 PCR 증폭하여 대장균에 클로닝하였다. Xylanase 유전자는 211 아미노산으로 구성된 단백질을 코드하며 633 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 xylanase는 glycosyl hydrolase family 11에 속하며 Paenibacillus의 xylanase와 85-89% 상동성을 보였다. Xylanase 유전자를 T7 promoter로 과잉발현한 결과 그 발현량이 높지 않았으며, 균체내 외에서 모두 효소활성이 관찰되었다. 재조합 대장균의 균체파쇄상등액을 사용하여 효소 반응특성을 조사한 결과 pH 5.5와 <TEX>$60^{\circ}C$</TEX>에서 최대 반응활성을 보였다. 한편 xylanase의 기질로 xylan과 xylooligosaccharides를 사용하였을 때 xylose와 xylotriose가 주된 최종 반응산물로 관찰되었으며 xylobiose는 분해하지 못하였으나 이보다 중합도가 큰 xylooligosaccharides는 분해하였다. A gene encoding the xylanase (XynA) predicted from partial genomic sequence of Paenibacillus woosongensis was cloned into Escherichia coli by PCR. This xynA gene consisted of 633 nucleotides, encoding a polypeptide of 211 amino acid residues. The deduced amino acid sequence exhibited 85-89% identity with those of several Paenibacillus xylanases, belonging to the glycosyl hydrolase family 11. As a results of expression of the structural gene by T7 promoter of a pET23a(+) expression vector, xylanase activity was higher in cell-free extract than culture filtrate of a recombinant Escherichia coli BL21(DE3) CodonPlus. However, the expression level of xylanase was not sufficient be detected by SDS-PAGE. The cell-free extract showed maximal xylanase activity at <TEX>$60^{\circ}C$</TEX> and pH 5.5. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylbiose.

Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides

Xylooligosaccharides have strong bifidogenic properties and are increasingly used as a prebiotic. Nonetheless, little is known about the degradation of these substrates by bifidobacteria. We characterized two recombinant β-xylosidases, XylB and XylC, with different substrate specificities from Bifidobacterium adolescentis. XylB is a novel β-xylosidase that belongs to the recently introduced glycoside hydrolase family 120. In contrast to most reported β-xylosidases, it shows only weak activity on xylobiose and prefers xylooligosaccharides with a degree of polymerization above two. The remaining xylobiose is efficiently hydrolyzed by the second B. adolescentis β-xylosidase, XylC, a glycoside hydrolase of family 43. Furthermore, XylB releases more xylose from arabinose-substituted xylooligosaccharides than XylC (30% and 20%, respectively). The different specificities of XylB, XylC, and the recently described reducing-end xylose-releasing exo-oligoxylanase RexA show how B. adolescentis can efficiently degrade prebiotic xylooligosaccharides.