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At least 109 records · Page 6Linked to original sources
PubMed2006

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase.

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.

Caulerpa↗
PubMed1997

Characterization of the xylanases from the new isolated thermophilic xylan-degrading Bacillus thermoleovorans strain K-3d and Bacillus flavothermus strain LB3A.

Three strictly aerobic strains (K-1, K-3d and K-4) were isolated from a hot-spring in Kobe, Japan, and a facultative anaerobic strain LB3A was isolated from sediments collected from the alkaline Lake Bogoria, Kenya. All strains were thermophilic and capable of growth on xylan. On the basis of morphological, physiological and phylogenetic studies the new aerobic isolates resemble the thermophilic species Bacillus thermoleovorans while the facultative anaerobic isolate LB3A resembles the facultative anaerobic thermophilic species Bacillus flavothermus. When grown on xylan as sole carbon source, all isolates produce thermoactive xylanases, Xylanases from strains K-3d and LB3A are active at temperatures between 40 and 90 degrees C and pH values between 5.0 and 9.0. Applying SDS-PAGE the crude xylanase complex of isolate K-3d was shown to be composed of two active bands, with molecular masses of 40 and 69 kDa. The crude xylanase complex of isolate LB3A, on the other hand, is composed of at least four activity bands with molecular masses ranging from 80 to 130 kDa. Due to the product pattern of xylan hydrolysis both enzymes are classified as endoxylanases. The xylanolytic enzyme system of isolate K-3d produces xylotriose, xylotetraose and larger xylooligosacharides, whereas the xylanases from isolate LB3A release xylotetraose as the major product of hydrolysis.

Bacillus↗
PubMed1992

Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca.

A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase were harvested at the end of fermentation and added to a xylan solution at 60 degrees C, thereby releasing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30 degrees C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestibility of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.

Base Sequence↗
PubMed1997

Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.

Acetylesterase↗
PubMed1983

Genes controlling xylan utilization by Bacillus subtilis.

Eight mutants of Bacillus subtilis deficient in xylan utilization were isolated and characterized genetically and biochemically. Each mutant was obtained independently after nitrosoguanidine mutagenesis. All of the analyzed mutations were shown to be linked. Reciprocal transformation crosses revealed the existence of two genes controlling xylan utilization which have been designated xynA and xynB. Available data have indicated that these two genes code for two xylan-degrading enzymes existing in the wild-type strains, an extracellular beta-xylanase (xynA) and a cell-associated beta-xylosidase (xynB).

Bacillus subtilis↗
PubMedDate not supplied

Cellulose and xylan degrading enzymes in Thecotheus pelletieri.

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.

Ascomycota↗
PubMed2006

Diurnal differences in the supply of glucomannans and xylans to innermost surface of cell walls at various developmental stages from cambium to mature xylem in Cryptomeria japonica.

We investigated the diurnal differences in the innermost surface of tracheid cell walls at various developmental stages from cambium to mature xylem. Cryptomeria japonica saplings were cultivated in a growth chamber with a light cycle set at 14 h of light and 10 h of darkness. Samples were collected from the saplings during both the light and dark periods. The innermost surface of cell walls was immunogold-labeled with anti-glucomannan or anti-xylan antiserum and was observed by field emission scanning electron microscopy. Diurnal differences in the aspect of the innermost surface of cell walls were seen only in S(2)-layer-forming tracheids; cellulose microfibrils were clearly evident during the light period, and amorphous material containing glucomannans and xylans was prevalent during the dark period. Cellulose microfibrils were present at the primary-wall formation and S(1)-layer-forming stages, and many warts were observed in the mature tracheids, regardless of the time of sampling. The densities of labeled glucomannans on the innermost surface of cell walls in S(1)- and S(2)-forming tracheids and of labeled xylans in S(2)-forming tracheids during the dark period were significantly higher than those during the light period. These results suggest a diurnal periodicity in the supply of cell wall matrix containing hemicellulose to the innermost surface of developing secondary walls.

Cell Wall↗
PubMed2005

Purification and properties of a low molecular weight 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.

A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.

Actinomycetales↗
PubMed2005

A family 10 Thermoascus aurantiacus xylanase utilizes arabinose decorations of xylan as significant substrate specificity determinants.

Xylan, which is a key component of the plant cell wall, consists of a backbone of beta-1,4-linked xylose residues that are decorated with arabinofuranose, acetyl, 4-O-methyl d-glucuronic acid and ferulate. The backbone of xylan is hydrolysed by endo-beta1,4-xylanases (xylanases); however, it is unclear whether the various side-chains of the polysaccharide are utilized by these enzymes as significant substrate specificity determinants. To address this question we have determined the crystal structure of a family 10 xylanase from Thermoascus aurantiacus, in complex with xylobiose containing an arabinofuranosyl-ferulate side-chain. We show that the distal glycone subsite of the enzyme makes extensive direct and indirect interactions with the arabinose side-chain, while the ferulate moiety is solvent-exposed. Consistent with the 3D structural data, the xylanase displays fourfold more activity against xylotriose in which the non-reducing moiety is linked to an arabinose side-chain, compared to the undecorated form of the oligosacchairde. These data indicate that the sugar decorations of xylans in the T.aurantiacus family 10 xylanase, rather than simply being accommodated, can be significant substrate specificity determinants.

Arabinose↗
PubMed2002

Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech.

The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1--> was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.

Acetylation↗
PubMed2003

Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan.

A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.

Bacterial Proteins↗
PubMed2001

Investigation on molar mass, solubility and enzymatic fragmentation of xylans by multi-detected SEC chromatography.

Four xylan samples from different origin were investigated, using a multi-detector, size exclusion, chromatographic system with two chromatographic column sets and mobile phases differing in the DMSO:water ratio. Molar mass distribution could be analysed best using a mobile phase of DMSO:water (90:10) with addition of 0.05 M LiBr, a system offering good solubilisation of the polymers and a proper chromatographic separation. SEC analysis in aqueous systems provided information on solubility and aggregation of xylans. A comparison of UV- and RI-signals in different systems gave further information on lignin impurities, which in some cases were involved in aggregation phenomena. Both analytical systems were applied to study the enzymatic fragmentation of xylans. Combining the information derived from the two systems can differentiate between the enzymatic degradation of the well-dissolved and the associated polymer fractions.

Bromides↗
PubMed2006

Improvement of binding activity of xylan-binding domain by amino acid substitution.

Xylanase J (XynJ) of alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme and consists of a glycoside hydrolase (GH) family 11 catalytic domain and an additional xylan-binding domain (XBD) belonging to carbohydrate-binding module (CBM) family 36. Random mutations were introduced into the XBD gene and the repertoire was cloned for display on the surface of filamentous phage. The mutant XBD with amino acid substitution T316I (Thr317 was replaced by Ile) showed higher xylan-binding activity compared to the wild-type XBD. Furthermore, hydrolyzing activity of XynJ toward insoluble xylan was also improved by introducing the mutation T316I.

Amino Acid Substitution↗
PubMed2002

A (1-->6)-linked beta-mannopyrananan, pseudonigeran, and a (1-->4)-linked beta-xylan, isolated from the lichenised basidiomycete Dictyonema glabratum.

Extraction of Dictyonema glabratum with hot 2% (w/v) aqueous KOH at 100 degrees C, followed by neutralisation and freeze-thawing, gave an insoluble glucan. The residue was further extracted by a similar process, but with hot 10% (w/v) aqueous KOH, furnishing a mixture of glucan, mannan and xylan. The mannan and xylan were obtained via precipitation of its copper complex with Fehling's solution, leaving the glucan in the supernatant. The insoluble complex was finally purified through gel permeation chromatography. Methylation analysis, one- and two-dimensional nuclear magnetic resonance examination showed the polysaccharides to be a (1-->3)-linked alpha-glucan (pseudonigeran) and a (1-->4)-linked beta-xylan, both not previously encountered in lichens, and a newly discovered (1-->6)-linked beta-mannan.

Glucans↗
PubMed1999

An alpha-L-arabinofuranosidase from Trichoderma reesei containing a noncatalytic xylan-binding domain.

L-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced alpha-L-arabinofuranosidase (alpha-AF) activity. An alpha-AF induced by L-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported alpha-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported alpha-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters for p-nitrophenyl-alpha-L-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported alpha-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

Amino Acid Sequence↗
PubMed2001

Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis.

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974-3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a beta-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.

Amino Acid Sequence↗
PubMed1984

Novel inducers of the xylan-degrading enzyme system of Cryptococcus albidus.

A series of compounds structurally related to xylan and 1,4-beta-xylobiose were tested as inducers of the xylan-degrading enzyme system of Cryptococcus albidus. Washed, glucose-grown cells were incubated with alpha- and beta-linked xylobioses, 4-O-beta-D-xylopyranosyl-L-arabinopyranose, 3-O-beta-D-xylopyranosyl-xylobiose, 6-O-beta-D-xylopyranosyl-cellobiose, cellobiose, and methyl beta-D-xylopyranoside. All alpha-xylobioses and cellobiose were inactive as inducers of the xylan-degrading enzyme system. Other compounds served as inducers of varying efficiency, depending on their concentration in the induction medium and the time of incubation of cells. The most rapid response of the cells, i.e., the shortest induction period of beta-xyloside permease, beta-xylosidase (EC 3.2.1.37), and beta-xylanase (EC 3.2.1.8), was observed with 1,4-beta-xylobiose, which was the most efficient inducer at low concentrations (0.1 to 0.2 mM). At higher concentrations (2 to 10 mM) and after long incubations, the highest enzyme yields were obtained with 1,2-beta-xylobiose. The results represent a new example of efficient induction of polysaccharide-degrading enzyme systems by positional isomers of dimers derived from the polysaccharide.

Cryptococcus↗
PubMed1985

Evaluation of xylan fermentation for the identification of Bacteroides ovatus and Bacteroides thetaiotaomicron.

The reliability of xylan fermentation in distinguishing Bacteroides ovatus from Bacteroides thetaiotaomicron was examined with strains previously identified by DNA homology studies or bacteriophage sensitivity testing or both. All of the 12 B. ovatus strains produced acid ranging from pH 5.0 to 5.6 in peptone-yeast-xylan broth; none of the 19 B. thetaiotaomicron strains produced acid, and the final pH was 6.0 or 6.1. Xylan fermentation and bacteriophage sensitivity-DNA homology appear to be equivalent in their ability to differentiate these two Bacteroides species.

Bacteriophage Typing↗