Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.
ABSTRACT: Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 ?-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.
Project description:A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30 °C, but Xyn10A retained 50% activity at 4 °C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-?-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose.
Project description:Background:The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity. Results:The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-d-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well. Conclusions:Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.
Project description:BACKGROUND:Xylanase is an important component of hemicellulase enzyme system. Since it plays an important role in the hydrolysis of hemicellulose into xylooligosaccharides (XOs), high thermostable xylanase has been the focus of much recent attention as powerful enzyme as well as in the field of biomass utilization. RESULTS:A xylanase gene (xyn10A) with 3,474 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum that encodes a protein containing 1,158 amino acid residues. Based on amino acid sequence homology, hydrophobic cluster and three dimensional structure analyses, it was attested that the xylanase belongs to the glycoside hydrolase (GH) families 10 with five carbohydrate binding domains. When the xylanase gene was cloned and expressed in Escherichia coli BL21 (DE3), the specific enzyme activity of xylanase produced by the recombinant strain was up to 145.8 U mg-1. The xylanase was optimally active at 95°C, pH 7.0. In addition, it exhibited high thermostability over broad range of pH 4.0-8.5 and temperature 55-90°C upon the addition of 5 mM Ca2+. Confirmed by Ion Chromatography System (ICS) analysis, the end products of the hydrolysis of beechwood xylan were xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose. CONCLUSIONS:The xylanase from T. thermarum is one of the hyperthermophilic xylanases that exhibits high thermostability, and thus, is a suitable candidate for generating XOs from cellulosic materials such as agricultural and forestry residues for the uses as prebiotics and precursors for further preparation of furfural and other chemicals.
Project description:In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-?-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n?=?2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl n (n?=?2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems.IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. ?-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.
Project description:The goal of this study was to enhance the production of xylooligosaccharides (XOs) and reduce the production of xylose. We investigated ?-xylosidases, which were key enzymes in the hydrolysis of xylan into xylose, in Trichoderma orientalis EU7-22. The binary vector pUR5750G/bxl::hph was constructed to knock out the ?-xyl1 gene (encoding ?-xylosidases) in T. orientalis EU7-22 by homologous integration, producing the mutant strain T. orientalis Bxyl-1. Xylanase activity for strain Bxyl-1 was 452.42 IU/mL, which increased by only 0.07% compared to that of parental strain EU7-22, whereas ?-xylosidase activity was 0.06 IU/mL, representing a 91.89% decrease. When xylanase (200 IU/g xylan), produced by T. orientalis EU7-22 and T. orientalis Bxyl-1, was used to hydrolyze beechwood xylan, in contrast to the parental strain, the XOs were enhanced by 83.27%, whereas xylose decreased by 45.80% after 36 h in T. orientalis Bxyl-1. Based on these results, T. orientalis Bxyl-1 has great potential for application in the production of XOs from lignocellulosic biomass.
Project description:1. Cell-free extracts from Epidinium ecaudatum (Crawley) hydrolysed the three hemicellulose fractions of pasture plants, but at different rates. 2. All of the constituent monosaccharides are released from the hemicellulose fractions, galactose and uronic acids being liberated at much slower rates than pentoses. 3. An arabinofuranosidase, which removes arabinose from highly branched arabinoxylan before the xylan chain can be hydrolysed, was isolated free from other pentosanases. 4. A xylanase hydrolysing xylan (by random cleavage) and xylodextrins of degree of polymerization (D.P.) > 3 to xylotriose and xylobiose was isolated free from other pentosanases. 5. A separate xylodextrinase hydrolysing (by random cleavage) xylodextrins of D.P. > 2 to xylobiose and xylose was also obtained; this enzyme did not hydrolyse xylan or xylobiose and the original extracts themselves possessed very weak xylobiase activity. 6. The epidinial extracts hydrolysed laminaribiose, laminarin, lichenin and cellodextrins of D.P. < 7 rapidly, cellobiose and gentiobiose slowly but cellulose not at all. 7. Polysaccharide glucose associated with plant linear B hemicellulose was liberated with cellobiose and possibly laminaribiose as intermediates. 8. The cellodextrinase hydrolysed cellopentaose initially to cellobiose plus cellotriose and is a distinctly different enzyme from the xylanase and xylodextrinase. 9. Extracts from Entodinium species and Eremoplastron bovis also hydrolysed all three types of plant hemicellose.
Project description:Bacterial strain Bacillus tequilensis BT21 isolated from marine sediments was found to produce extracellular xylanase. The xynBT21 gene encoding xylanase enzyme was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acid residues with calculated molecular mass of 23.3 kDa. Puri?ed recombinant xylanase had optimum activity at 60 °C and pH=6. The enzyme was highly stable in alkaline pH, at pH=7 it remained 100% active for 24 h, while its activity increased at pH=8 and 9 during incubation. B. tequilensis BT21 xylanase had alkaline pI of 9.4 and belongs to glycosyl hydrolase family 11. The mode of action of XynBT21 on beechwood xylan and xylooligosaccharides was studied. It hydrolysed xylooligosaccharides and beechwood xylan yielding mainly xylobiose (X2) with a small amount of xylose (X1), indicating that XynBT21 was probably an endo-acting xylanase. Enzymatic hydrolysis using wheat bran as a substrate revealed that xylanase reported here has the potential to produce xylobiose from wheat bran. Xylooligosaccharides, especially xylobiose, have strong bifidogenic properties and are increasingly used as a prebiotic. This is the ?rst report that describes this novel xylanase enzyme from marine B. tequilensis BT21 used for the release of xylobiose from wheat bran.
Project description:Background:Prehydrolyzate, which is from the prehydrolysis process in dissolving pulps industry, contains various sugar-derived and lignin compounds such as xylooligosaccharides (XOS), gluco-oligosaccharides, xylose, glucose, and soluble lignin (S-L). The XOS has several beneficial effects on human physiology. XOS and S-L in prehydrolyzate are difficult to efficiently fractionate due to their similar molecular weights and water solubility. In this work, we proposed a sustainable and green process using polystyrene divinylbenzene (PS-DVB) resin to simultaneously separate and recover XOS and S-L. Enzymatic hydrolysis with endo-1,4-β-xylanase and fermentation with P. stipites were sequentially applied to purify XOS to minimize xylose content as well as amplify contents of xylobiose and xylotriose. In addition, 2D-HSQC NMR was used to analyze the structural characteristics of XOS and S-L. Furthermore, the biological abilities of antioxidants and prebiotics of these fractions were investigated by scavenging radicals and cultivating intestinally beneficial bacterias, respectively. Results:Results showed that PS-DVB resin could simultaneously separate XOS and solubilized lignin with excellent yields of 93.2% and 85.3%, respectively. The obtained XOS after being purified by enzymatic hydrolysis and fermentation contained 57.7% of xylobiose and xylotriose. 10.4% amount of inherent xylan was found in the S-L fraction obtained by PS-DVB resin separation. 2D-HSQC NMR revealed that lignin carbohydrate complexes existed in both XOS and S-L as covalent linkages between lignin and 4-O-methylglucuronoarabinoxylan. The biological application results showed that the antioxidant capacity of S-L was stronger than XOS, while XOS was superior in promoting growth of intestinal Bifidobacteria adolescentis and stimulating production of short-chain fatty acids by Lactobacillus acidophilus. Conclusions:The proposed strategy of sequentially combining hydrophobic resin separation, enzymatic hydrolysis, and fermentation was successfully demonstrated and resulted in simultaneous production of high-quality XOS and solubilized lignin. These biomass-derived products in prehydrolyzate can be regarded as value-adding prebiotics and antioxidants.
Project description:The preparation of oligosaccharides via xylan hydrolysis is an effective way to add value to hemicellulosic material of agricultural waste. The bacterial strain Streptomyces L10608, isolated from soil, contains genes encoding xylanases of glucoside hydrolase family 10/11 (GH10/11), and these have been cloned to catalyze the production of xylooligosaccharide (XOS). To improve the XOS proportion of hydrolysates produced by xylanase, four amino acid residues were substituted by site-directed mutagenesis, and the mutant genes were overexpressed in Escherichia coli. Mutations replaced the codons encoding Asn214 (+2) and Asn86 (-2) by Ala and removed the Ricin B-lectin domain in GH10-xyn, and mutants Y115A (-2) and Y123A (-2) were produced for GH11-xyn. Interestingly, GH10-N86Q had significantly increased hydrolysis of XOS and almost eliminated xylose (X1) to <2.5%, indicating that the -2 binding site of GH10-xyn of L10608 is required for binding with xylotriose (X3). The hydrolytic activity of GH10-N86Q was increased approximately 1.25-fold using beechwood xylan as a substrate and had high affinity for the substrate with a low Km of about 1.85 mg·mL-1. Otherwise, there were no significant differences in enzymatic properties between GH10-N86Q and GH10-xyn. These mutants offer great potential for modification of xylanase with desired XOS hydrolysis.
Project description:This study investigated pilot-scale production of xylo-oligosaccharides (XOS) and fermentable sugars from Miscanthus using steam explosion (SE) pretreatment. SE conditions (200?°C; 15?bar; 10?min) led to XOS yields up to 52 % (w/w of initial xylan) in the hydrolysate. Liquid chromatography-mass spectrometry demonstrated that the solubilised XOS contained bound acetyl- and hydroxycinnamate residues, physicochemical properties known for high prebiotic effects and anti-oxidant activity in nutraceutical foods. Enzymatic hydrolysis of XOS-rich hydrolysate with commercial endo-xylanases resulted in xylobiose yields of 380 to 500?g/kg of initial xylan in the biomass after only 4?h, equivalent to ~74 to 90 % conversion of XOS into xylobiose. Fermentable glucose yields from enzymatic hydrolysis of solid residues were 8 to 9-fold higher than for untreated material. In view of an integrated biorefinery, we demonstrate the potential for efficient utilisation of Miscanthus for the production of renewable sources, including biochemicals and biofuels.