Characterization of the xylan-degrading microbial community from human faeces

In humans, plant cell wall polysaccharides represent an important source of dietary fibres that are digested by gut microorganisms. Despite the extensive degradation of xylan in the colon, the population structure and the taxonomy of the predominant bacteria involved in degradation of this polysaccharide have not been extensively explored. The objective of our study was to characterize the xylanolytic microbial community from human faeces, using xylan from different botanic origins. The xylanolytic population was enumerated at high level in all faecal samples studied. The predominant xylanolytic organisms further isolated (20 strains) were assigned to Roseburia and Bacteroides species. Some Bacteroides isolates corresponded to the two newly described species Bacteroides intestinalis and Bacteroides dorei. Other isolates were closely related to Bacteroides sp. nov., a cellulolytic bacterium recently isolated from human faeces. The remaining Bacteroides strains could be considered to belong to a new species of this genus. Roseburia isolates could be assigned to the species Roseburia intestinalis. The xylanase activity of the Bacteroides and Roseburia isolates was found to be higher than that of other gut xylanolytic species previously identified. Our results provide new insights to the diversity and activity of the human gut xylanolytic community. Four new xylan-degrading Bacteroides species were identified and the xylanolytic capacity of R. intestinalis was further shown.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

H2 and acetate transfers during xylan fermentation between a butyrate-producing xylanolytic species and hydrogenotrophic microorganisms from the human gut

The aim of this work was to investigate in vitro interrelationships during xylan fermentation between an H2 and butyrate-producing xylanolytic species recently isolated in our laboratory from human faeces and identified as Roseburia intestinalis and the H2-utilizing acetogen Ruminococcus hydrogenotrophicus or the methanogen Methanobrevibacter smithii. H2 transfer between M. smithii or Ru. hydrogenotrophicus and the xylanolytic species was evidenced, confirming the great potential of these H2-consuming microorganisms to reutilize fermentative H2 during fibre fermentation in the gut. In addition, acetate transfer was demonstrated between the xylanolytic Roseburia sp. and the acetogenic species, both metabolites transfers leading to butyric fermentation of oat xylan without production of H2.     

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml⁻¹, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml⁻¹ was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.     

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community’s structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.     

A role of xylanase,alpha-L-arabinofuranosidase,and xylosidase in xylan degradation

Renewable natural resources such as xylans are abundant in many agricultural wastes. Penicillium sp. AHT-1 is a strong producer of xylanolytic enzymes. The sequential activities of its xylanase, alpha-L-arabinofuranosidase, and beta-xylosidase on model hemicellulose oat-spelt xylan was investigated. Optimum production of the enzymes was found in culture containing oat-spelt xylan at 30 degrees C and initial pH 7.0 after 6 days. The enzymes were partially purified by ammonium sulphate fractionation and anion-exchange chromatography on DEAE-Toyopearl 650 S. The apparent molecular mass was 21 kDa, and the protein displayed an "endo" mode of action. The xylanase exhibited glycotansferase activity. It synthesized higher oligosaccharides from the initial substrates, and xylotriose was the shortest unit of substrate transglycosylated. Xylanolytic enzymes (enzyme mixture) produced by this Penicillium sp. interacted cooperatively and sequentially in the hydrolysis of oat-spelt xylan in the following order: alpha-L-arabinofuranosidase --> xylanase --> beta-xylosidase. All three enzymes exhibited optimal activity under the same conditions (temperature, pH, cultivation), indicating that they alone are sufficient to completely depolymerize the test xylan. Results indicate that the xylanolytic enzyme mixture of Penicillium sp. AHT-1 could be useful for bioconversion of xylan-rich plant wastes to value-added products.     

Studies on Carboxymethyl Cellulase and Xylanase Activities of Anaerobic Fungal Isolate CR4 from the Bovine Rumen

An anaerobic fungal isolate, CR4, was isolated from the bovine rumen. The DNA sequence of internal transcribed spacer region 1 showed that CR4 belonged to the genus Caecocmyces. The dry matter digestibility of timothy hay by anaerobic fungal isolate CR4 was determined. The effects of carbohydrate growth substrates on carboxymethyl cellulase (CMCase) and xylanase activities also were examined. The extent of dry matter digestibility of timothy hay was 31% at 6 days’ incubation. The highest specific activity of CMCase in the culture supernatant (SN) fraction was observed in xylose culture. The activity of CMCase was not detected in the SN fraction of cellobiose and xylan or in the cell-bound fraction of all growth substrates. The highest specific activity of xylanase in the SN fraction was observed in glucose culture. These results suggest that fiber-degrading enzyme activities were affected by growth substrates and that CR4 is xylanolytic. Zymogram analysis showed that CR4 produces three CMCases of molecular mass (95, 89, and 64 kDa) and three xylanases of molecular mass (82, 73, and 66 kDa). This is the first demonstration showing the molecular mass of fiber-degrading enzymes of Caecomyces.     

Multiple endoxylanases of Butyrivibrio sp

Butyrivibrio sp. Mz 5 with a high xylanolytic activity was isolated. Four major xylanases were detected in the cell-associated fraction using the zymogram technique. The xylanolytic activity was inducible with the oat spelts xylan; two endoxylanases (51 and 145 kDa) were formed constitutively. The bulk of the xylanolytic activity was cell-bound and growth-phase dependent; the maximum activity in the cell-associated fraction was achieved after 16 h of incubation. The highest xylanolytic activity was determined in a medium with 0.5% oat spelts xylan. Under optimum conditions (the highest xylanolytic activity produced), the two cell-bound xylanases (51 and 58 kDa) were isolated by anion exchange chromatography on CIM DEAE 8 tubes attached to a MPLC system, and gel filtration.     

Synthesis and regulation of D-xylanase from Cellulomonas flavigena wild type and a mutant

The xylanolytic system from Cellulomonas flavigena was enhanced by adding cellulose to the growth medium. The Solka floc:xylan (60:40 w/w) mixture induced xylanase synthesis by more than 3-fold over that induced by growing C. flavigena, wild type and its mutant PN-120 on pure xylan. The hydrolysis pattern of sugar cane bagasse and xylan indicated the presence of debranching endo-;-xylanase activity.     

Xylanase production using inexpensive agricultural wastes and its partial characterization from a halophilic <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TPSV 101

A halophilic and alkali-tolerant Chromohalobacter sp. TPSV 101 with an ability to produce extracellular halophilic, alkali-tolerant and moderately thermostable xylanase was isolated from solar salterns. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. The culture conditions for higher xylanase production were optimized with respect to NaCl, pH, temperature, substrates and metal ions and additives. Maximum xylanase production was achieved in the medium with 20% NaCl, pH-9.0 at 40°C supplemented with 1% (w/v) sugarcane bagasse and 0.5% feather hydrolysate as carbon and nitrogen sources. Sugarcane bagasse (250 U/ml) and wheat bran (190 U/ml) were the best inducer of xylanase when used as carbon source as compared to xylan (61 U/ml). The xylanase that was partially purified by protein concentrator had a molecular mass of 15 kDa approximately. The xylanase from Chromohalobacter sp. TPSV 101 was active at pH 9.0 and required 20% NaCl for optimal xylanolytic activity and was active over a broad range of temperature 40–80°C with 65°C as optimum. The early stage hydrolysis products of sugarcane bagasse were xylose and xylobiose, after longer periods of incubation only xylose was detected.     

Cellulase-free xylanase by thermophilic fungi: a comparison of xylanase production by two Thermomyces lanuginosus strains

Thermomyces lanuginosus strains RT9 and MH4 were studied to find favourable cultivation conditions and to compare their abilities to produce xylanolytic enzymes in three media on different substrates at 50° C or 55° C under shake-culture conditions. Both organisms produced xylanases free of cellulase at widely different levels in all cultivation conditions employed. Wheat bran, corn cobs and xylan induced xylanases in increasing order of producing with both cultures. T. lanuginosus RT9 demonstrated the highest xylanase production in all cultivation conditions but with lower soluble protein, reducing sugar, -xylosidase and debranching enzymes levels (arabinosidase, acetylxylanesterase, mannanase) when compared to T. lanuginosus MH4. The study reveals that xylanase production was highly influenced by nitrogen sources and their concentrations and by the initial pH in the cultures. The two strains may therefore be unique, when technical application is considered in terms of the quantity and quality of the xylanolytic enzymes produced.     

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes.The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan.When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.     

Xylanase production by a new alkali-tolerant isolate of Bacillus

The xylanolytic system of an alkali-tolerant Bacillus sp. consists of several xylanases ranging from 22 to 120 kDa and pI values from 7.0 to 9.0. Crude xylanase retained 72% of initial activity after 5 h at pH 9.0 and 45°C. Xylanase production was induced by xylose and xylan and was maximum at 42°C and pH 7.8. Crude xylanase released xylotriose and xylotetraose as main products of xylan hydrolysis. Xylose was not detected. © Rapid Science Ltd. 1998     

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-d-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-d-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-d-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-d-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-d-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and/or its hydrolysis products (xylo-oligosaccharides and xylose). Xylanolytic yeasts are able to secrete xylanolytic enzymes mainly when induced by xylan and present different strategies (intra- and/or extracellular hydrolysis) for the metabolism of xylo-oligosaccharides. Some of the unique xylanolytic traits identified here should be further explored for their applicability in specific biotechnological processes.     

The identification,purification, and characterization of STXF10 expressed in <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis> NTU-88

Multiple xylanolytic enzymes of Streptomyces thermonitrificans NTU-88 were induced by oat-spelt xylan and separated by two-dimensional polyacrylamide and zymogram gels. Nineteen clear spots differed in pI and molecular weight values were found on the zymogram, and only spot one was seen on the corresponding silver-stained gel. These results revealed that multiple xylanases were secreted when S. thermonitrificans NTU-88 was induced and the spot (STXF10), identified as being a glycosyl hydrolase family 10 xylanase, was the predominant one among xylanases. STXF10 showed a tolerance for high temperatures and broad pH ranges and high affinity and hydrolysis efficiency for xylans. Furthermore, it also featured the minor ability to degrade different lignocellulosic substrates. Although S. thermonitrificans NTU-88 possesses multiple xylanases, our results suggest that the major form of xylanase might be selectively and specifically induced depending on the type of substrate to which the microorganism is exposed.     

Streptococcus shiloi andStreptococcus difficile: Two new streptococcal species causing a meningoencephalitis in fish

The cellulolytic rumen bacteriumRuminococcus flavefaciens 17 was found to produce multiple xylanases ranging in apparent molecular weight from 55 to 200 kDa. A 55 kDa xylanase showed constitutive synthesis, but formation of the larger enzymes was increased in cultures grown with avicel, straw, or xylan, compared with cellobiose, as the energy source. At least six xylanases were detected in cultures grown with oat straw or oat xylan. Polyclonal antibodies were raised against the amino (A) or carboxy terminal (C) domains of the bifunctional XYNA product of the clonedR. flavefaciens xynA gene. Both antibody preparations recognized several xylanases larger than 80 kDa fromR. flavefaciens cells grown with avicel, straw, or xylan, indicating the production of multiple, antigenically related enzymes during growth on these substrates. Neither antibody preparation recognized the constitutive 55-kDa xylanase.     

Hydrolysis of xylans by a thermostable hybrid xylanase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli-expressed a hybrid xylanase, Btx, encoded by a designed hybrid xylanase gene btx was purified. The molecular mass of the enzyme was estimated to be 22 kDa. The K _m and k _cat values for Btx were 1.9 mg/ml and 140 s⁻¹, respectively. It hydrolyzed xylan principally to xylobiose and xylotriose, and was functionally similar to family 11 xylanases. As some differences were found in the hydrolytic products between birchwood xylan and wheat bran insoluble xylan, the xylan binding domains in xylanase Btx must have different effects on soluble and insoluble xylan.     

The antimicrobial action of chitosan,low molar mass chitosan,and chitooligosaccharides on human colonic bacteria

Antibacterial effect of chitooligosaccharides (COS) and low molar mass chitosans (LMWC) is considered as one of the most important characteristics of chitosan (CS) hydrolysates. Here, we show the in vitro effect of different COS, LMWC, and CS on representative anaerobic bacteria isolated from human colon as a possibility of targeting modification of colonic microflora composition by supplementation of dietary CS products by humans. Specific growth rate of seven selected nonpathogenic anaerobic bacterial strains (Clostridium paraputrificum, Clostridium beijerinckii, Roseburia intestinalis, Bacteroides vulgatus, Bacteriodes thetaiotaomicron, Faecalibacterium prausnitzii and Blautia coccoides) was determined in the presence of 0.25 and 0.5% COS (2, 3, and 6 kDa), 0.025 and 0.05% of LMWC (10 and 16 kDa), and 0.025 and 0.1% of CS in vitro. The growth rate decreased in all strains in the presence of COS and LMWC in higher concentrations in comparison to control incubations. A relatively higher resistance to CS hydrolyzates was detected in R. intestinalis and F. prausnitzii, and more susceptible were bacteria belonging to Bacteoides sp. and Clostridium sp. The antimicrobial activity, minimum inhibitory concentrations (MIC), and minimal bactericidal concentrations (MBC) were determined. The antimicrobial activity increased with the degree of polymerization (DP). MIC ranged from 0.25 to 4.5% in dependence on bacterial strain and DP of CS/LMWC. MBC also decreased with DP. The most effective antimicrobial action was detected in LMWC with 16 kDa and CS. Weak antimicrobial activity was found in COS with small molecules (2 and 3 kDa).     

Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1

An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe²⁺, Ca²⁺, and Mg²⁺ greatly increased the xylanase activity, whereas Mn²⁺ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.     

Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked β-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of β-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. β-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also. Received: 16 May 1997 / Received revision: 12 September 1997 / Accepted: 19 September 1997