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1.
Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAXn), a beta-1,4 xylan in which 10% to 20% of the xylose residues are substituted with alpha-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAXn. A modular xylanase (XynA1) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA1 (XynA1 CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAXn as substrate, XynA1 CD generated xylobiose and aldotetrauronate (MeGAX3) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAXn, as well as decreased growth rate and yield with XynA1 CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA1 catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAXn to alternative fuels and biobased products. 相似文献
2.
Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors. 相似文献
3.
Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX n), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX n and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX 3, the aldotetrauronate 4- O-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX n and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element ( cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products. 相似文献
4.
Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica. 相似文献
5.
Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4- O-methylglucuronoxylan (MeGX n) as directed by the position along the xylan chain of an α-1,2-linked 4- O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGX n by these enzymes results in singly substituted aldouronates having a 4- O-methylglucuronate moiety linked to a xylose penultimate from the reducing terminal xylose and some number of xylose residues toward the nonreducing terminus. This novel mode of action distinguishes GH30 xylanases from the more common xylanase families that cleave MeGX n in accessible regions. To help understand this unique biochemical function, we have determined the structure of XynC in its native and ligand-bound forms. XynC structure models derived from diffraction data of XynC crystal soaks with the simple sugar glucuronate (GA) and the tetrameric sugar 4- O-methyl-aldotetrauronate resulted in models containing GA and 4- O-methyl-aldotriuronate, respectively. Each is observed in two locations within XynC surface openings. Ligand coordination occurs within the XynC catalytic substrate binding cleft and on the structurally fused side β-domain, demonstrating a substrate targeting role for this putative carbohydrate binding module. Structural data reveal that GA acts as a primary functional appendage for recognition and hydrolysis of the MeGX n polymer by the protein. This work compares the structure of XynC with a previously reported homologous enzyme, XynA, from Erwinia chrysanthemi and analyzes the ligand binding sites. Our results identify the molecular interactions that define the unique function of XynC and homologous GH30 enzymes. 相似文献
6.
An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase. 相似文献
7.
Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan. 相似文献
8.
An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase. 相似文献
10.
Paenibacillus sp. JDR-2 (Pjdr2) has been studied as a model for development of bacterial biocatalysts for efficient processing of xylans, methylglucuronoxylan, and methylglucuronoarabinoxylan, the predominant hemicellulosic polysaccharides found in dicots and monocots, respectively. Pjdr2 produces a cell-associated GH10 endoxylanase (Xyn10A1) that catalyzes depolymerization of xylans to xylobiose, xylotriose, and methylglucuronoxylotriose with methylglucuronate-linked α-1,2 to the nonreducing terminal xylose. A GH10/GH67 xylan utilization regulon includes genes encoding an extracellular cell-associated Xyn10A1 endoxylanase and an intracellular GH67 α-glucuronidase active on methylglucuronoxylotriose generated by Xyn10A1 but without activity on methylglucuronoxylotetraose generated by a GH11 endoxylanase. The sequenced genome of Pjdr2 contains three paralogous genes potentially encoding GH115 α-glucuronidases found in certain bacteria and fungi. One of these, Pjdr2_5977, shows enhanced expression during growth on xylans along with Pjdr2_4664 encoding a GH11 endoxylanase. Here, we show that Pjdr2_5977 encodes a GH115 α-glucuronidase, Agu115A, with maximal activity on the aldouronate methylglucuronoxylotetraose selectively generated by a GH11 endoxylanase Xyn11 encoded by Pjdr2_4664. Growth of Pjdr2 on this methylglucuronoxylotetraose supports a process for Xyn11-mediated extracellular depolymerization of methylglucuronoxylan and Agu115A-mediated intracellular deglycosylation as an alternative to the GH10/GH67 system previously defined in this bacterium. A recombinantly expressed enzyme encoded by the Pjdr2 agu115A gene catalyzes removal of 4-O-methylglucuronate residues α-1,2 linked to internal xylose residues in oligoxylosides generated by GH11 and GH30 xylanases and releases methylglucuronate from polymeric methylglucuronoxylan. The GH115 α-glucuronidase from Pjdr2 extends the discovery of this activity to members of the phylum Firmicutes and contributes to a novel system for bioprocessing hemicelluloses. 相似文献
11.
Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and displayed on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylanase inhibitor TAXI, each time preceded by an incubation step at elevated temperature. DNA sequence analysis of enriched phagemid panning isolates allowed identification of mutations conferring enhanced thermal stability. In particular, substitutions T44C, T44Y, F48C, T87D, and Y94C were retained, and their thermostabilizing effect was confirmed by testing site-directed XynA variants. None of these mutations was identified in earlier endoxylanase engineering studies. Each single mutation increased the half-inactivation temperature by 2-3 °C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges. 相似文献
12.
The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass. 相似文献
13.
Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and xynC genes, respectively. Both of these enzymes have been defined with respect to structure and function. In this study, the effects of deletion of the xynA and xynC genes, individually and in combination, were evaluated for xylan utilization and formation of acidic xylooligosaccharides. Parent strain 168 depolymerizes methylglucuronoxylans (MeGX n), releasing the xylobiose and xylotriose utilized for growth and accumulating the aldouronate methylglucuronoxylotriose (MeGX 3) with some methylglucuronoxylotetraose (MeGX 4). The combined GH11 and GH30 activities process the products generated by their respective actions on MeGX n to release a maximal amount of neutral xylooligosaccharides for assimilation and growth, at the same time forming MeGX 3 in which the internal xylose is substituted with methylglucuronate (MeG). Deletion of xynA results in the accumulation of β-1,4-xylooligosaccharides with degrees of polymerization ranging from 4 to 18 and an average degree of substitution of 1 in 7.2, each with a single MeG linked α-1,2 to the xylose penultimate to the xylose at the reducing terminus. Deletion of the xynC gene results in the accumulation of aldouronates comprised of 4 or more xylose residues in which the MeG may be linked α-1,2 to the xylose penultimate to the nonreducing xylose. These B. subtilis lines may be used for the production of acidic xylooligosaccharides with applications in human and veterinary medicine. 相似文献
14.
D-Ribose is a functional five-carbon sugar, which has been used for the commercial production of riboflavin. Mechanisms of d-ribose biosynthesis from xylose were investigated in the genetically engineered Bacillus subtilis JY200 with a deficiency in transketolase. A transketolase gene (tkt) disruption cassette in plasmid pUNKC was introduced into the chromosomal tkt gene in the wild type B. subtilis 168. Analysis of culture broth by thin layer chromatography confirmed that the disruption of tkt allowed B. subtilis JY200 to produce d-ribose. In a batch culture of B. subtilis JY200, a loss of cell viability was observed after glucose depletion. Fed-batch cultivation by feeding 400 gl(-1) glucose solution as a co-substrate was carried out to supply energy to xylose metabolism and to maintain cell viability throughout cultivation. Fed-batch cultivation of B. subtilis JY200 in a complex medium containing 11 gl(-1) xylose and 5 gl(-1) glucose initially gave the best result of 10.1 gl(-1)D-ribose concentration, 0.24 gg(-1)D-ribose yield and 0.29 gl(-1)h(-1) productivity, corresponding to 40-, 5- and 12-fold increases compared with those in the batch culture. A kinetic study of D-ribose production in fed-batch cultivations of B. subtilis JY200 suggested that xylose uptake might be critical to maximize D-ribose biosynthesis from xylose. 相似文献
15.
It has been known that wild type Bacillus subtilis cannot grow rapidly in a minimal medium containing xylose as a sole carbon source because it does not have a xylose-specific transporter. In this study, the arabinose:H(+) symporter, AraE protein from B. subtilis was expressed in B. subtilis 168 in order to transport xylose efficiently. The AraE expression cassette was constructed to contain the xylose-inducible xylA promoter, araE gene and fba terminator, and integrated into the chromosomal amyE gene in B. subtilis 168. Batch cultures in a defined medium with xylose only or a mixture of xylose and glucose showed that expression of AraE led to fast and complete consumption of initially added xylose and hence a considerable increase in cell growth of the recombinant B. subtilis JY123 expressing AraE. Considering the systematic analysis of cell growth, sugar consumption, respiratory quotient and xylulokinase activity, it was certain that AraE protein could transport xylose into B. subtilis efficiently. 相似文献
16.
The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen. 相似文献
18.
The relative activity of an endoxylanase towards water-unextractable (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, referred to as endoxylanase substrate selectivity, impacts the enzyme functionality in cereal-based biotechnological processes such as bread-making and gluten starch separation. A set of six endoxylanases representing a range of substrate selectivities as determined by a screening method using chromophoric substrates [Anal. Biochem.2003, 319, 73-77] was used to examine the impact of such selectivity on changes in structural characteristics of wheat WU-AX and WE-AX upon enzymic hydrolysis. While WE-AX degradation by the selected endoxylanases was very comparable with respect to apparent molecular mass (MM) profiles and arabinose to xylose ratio of the hydrolysates formed, WU-AX solubilisation and subsequent degradation of solubilised fragments gave rise to widely varying MM profiles, depending on the substrate selectivity of the enzymes. Enzymes with high selectivity towards WU-AX de facto generated higher MM fragments from WU-AX than enzymes with low selectivity. The arabinose to xylose ratios of solubilised fragments were independent of the degree of solubilisation. 相似文献
19.
Abstract Bacillus subtilis 168 is unable to effectively utilize xylose as sole carbon source. We demonstrate here that this strain cannot actively transport xylose into the cell. After leaving B. subtilis 168 for a few days on minimal plates with xylose as sole carbon source large colonies arise with a frequency of 1 × 10 −6/cell. These mutants grow well on xylose and efficiently take up that sugar. This new property is not inducible by xylose, indicating that the mutation is neither in the xyl nor in the xyn operon. 相似文献
20.
Xylan has a main chain consisting of β-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the − 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the − 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects. 相似文献
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