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1.
Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (> or = X3) to release X1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which alpha-X2F was hydrolyzed to X2 and HF in the presence of X1 as an acceptor molecule. However, the transglycosidation product (X3) was not detectable in the reaction. To convert reducing end xylose-releasing exooligoxylanase to glycosynthase, derivatives with mutations in the catalytic base (Asp-263) were constructed by saturation random mutagenesis. Nine amino acid residue mutants (Asp-263 to Gly, Ala, Val, Thr, Leu, Asn, Cys, Pro, or Ser) were found to possess glycosynthase activity forming X3 from alpha-X2F and X1. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of alpha-X2F. The D263C mutant showed 10-fold lower hydrolytic activity than D263N, resulting in the highest yield of X3. X2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X3 formed by condensation was hydrolyzed before its release from the enzyme. To acquire glycosynthase activity from inverting enzymes, it is important to minimize the decrease in F(-)-releasing activity while maximizing the decrease in the hydrolytic activity. The present study expands the possibility of conversion of glycosynthases from inverting enzymes.  相似文献   

2.
An arginine residue in loop 4 connecting beta strand 4 and alpha-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K(m) and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.  相似文献   

3.
Although many xylanases are widely used in the baking industry, only one glycoside hydrolase family 10 (GH 10) xylanase has previously been reported to be effective in baking. In this study, we compared the effectiveness of two GH 10 xylanases, psychrophilic XynA from Glaciecola mesophila and mesophilic EX1 from Trichoderma pseudokoningii, in bread making. The optimal dosages needed to improve wheat flour dough and bread quality were 270-U/kg flour for EX1 and 0.9-U/kg flour for XynA. At their optimal dosage, both XynA and EX1 had significant dough-softening ability, resulting in a 50% reduction in Brabender units. XynA was more effective than EX1 in reducing the time to reach maximum consistency. XynA and EX1 showed similar effects in improving the bread volume (~30% increase). EX1 was more effective in reducing the initial crumb firmness. Although both enzymes exhibited similar anti-staling effects on the bread, based on a decrease in the bread firmness, XynA had a greater effect on reducing the firming rate, and EX1 showed an enhanced reduction in the initial firmness. These results show that these two GH 10 xylanases have unique advantages in improving dough and bread quality and indicate their potential in bread making.  相似文献   

4.
The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.  相似文献   

5.
The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.  相似文献   

6.
Xylanases are of widespread importance in several food and non-food biotechnological applications. They degrade heteroxylans, a structurally heterogeneous group of plant cell wall polysaccharides, and other important components in various industrial processes. Because of the highly complex structures of heteroxylans, efficient utilization of xylanases in these processes requires an in-depth knowledge of their substrate specificity. A significant number of studies on the three-dimensional structures of xylanases from different glycoside hydrolase (GH) families in complex with the substrate provided insight into the different mechanisms and strategies by which xylanases bind and hydrolyze structurally different heteroxylans and xylo-oligosaccharides (XOS). Combined with reports on the hydrolytic activities of xylanases on decorated XOS and heteroxylans, major advances have been made in our understanding of the link between the three-dimensional structures and the substrate specificities of these enzymes. In this review, authors gave a concise overview of the structure–function relationship of xylanases from GH5, 8, 10, and 11. The structural basis for inter- and intrafamily variation in xylanase substrate specificity was discussed as are the implications for heteroxylan degradation.  相似文献   

7.
8.
Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.  相似文献   

9.
Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.  相似文献   

10.
The potential of glycoside hydrolase family (GH) 8 xylanases in biotechnological applications is virtually unexplored. Therefore, the substrate preference and hydrolysis product profiles of two GH8 xylanases were evaluated to investigate their activities and substrate specificities. A GH8 xylanase from an uncultured bacterium (rXyn8) shows endo action but very selectively releases xylotriose from its substrates. It has a higher activity than the Pseudoalteromonas haloplanktis GH8 endo-xylanase (PhXyl) on xylononaose and smaller xylo-oligosaccharides. PhXyl preferably degrades xylan substrates with a high degree of polymerization. It is sterically more hindered by arabinose substituents than rXyn8, producing larger end hydrolysis products. The specificities of rXyn8 and PhXyl differ completely from these of the previously described GH8 xylanases from Bifidobacterium adolescentis (BaRexA) and Bacillus halodurans (BhRex). As reducing-end xylose-releasing exo-oligoxylanases, they selectively release xylose from the reducing end of small xylo-oligosaccharides. The findings of this study show that GH8 xylanases have a narrow substrate specificity, but also one that strongly varies between family members and is distinct from that of GH10 and GH11 xylanases. Structural comparison of rXyn8, PhXyl, BaRexA, and BhRex showed that subtle amino acid changes in the glycon as well as the aglycon subsites probably form the basis of the observed differences between GH8 xylanases. GH8 xylanases, therefore, are an interesting group of enzymes, with potential towards engineering and applications.  相似文献   

11.
12.
G Wang  K Meng  H Luo  Y Wang  H Huang  P Shi  P Yang  Z Zhang  B Yao 《PloS one》2012,7(8):e43480

Background

Xylan is one of the most abundant biopolymers on Earth. Its degradation is mediated primarily by microbial xylanase in nature. To explore the diversity and distribution patterns of xylanase genes in soils, samples of five soil types with different physicochemical characters were analyzed.

Methodology/Principal Findings

Partial xylanase genes of glycoside hydrolase (GH) family 10 were recovered following direct DNA extraction from soil, PCR amplification and cloning. Combined with our previous study, a total of 1084 gene fragments were obtained, representing 366 OTUs. More than half of the OTUs were novel (identities of <65% with known xylanases) and had no close relatives based on phylogenetic analyses. Xylanase genes from all the soil environments were mainly distributed in Bacteroidetes, Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Dictyoglomi and some fungi. Although identical sequences were found in several sites, habitat-specific patterns appeared to be important, and geochemical factors such as pH and oxygen content significantly influenced the compositions of xylan-degrading microbial communities.

Conclusion/Significance

These results provide insight into the GH 10 xylanases in various soil environments and reveal that xylan-degrading microbial communities are environment specific with diverse and abundant populations.  相似文献   

13.
Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that rapidly digests crystalline cellulose in a contact-dependent manner. The different roles of various predicted glycoside hydrolases and the detailed mechanism used by C. hutchinsonii in cellulose digestion are, however, not known. In this study, an endoglucanase belonging to glycoside hydrolase family 5 (GH5) named as ChCel5A was isolated from the outer membrane of C. hutchinsonii. The catalytic domain of ChCel5A exhibited typical endoglucanase activity and was capable of hydrolyzing insoluble cellulose with cellobiose and cellotriose as the predominant digestion products. Site-directed mutagenesis identified two aromatic amino acids in ChCle5A, W61 and W308, that dramatically decreased its hydrolytic activity toward filter paper while causing only a slight decrease in carboxymethylcellulase (CMCase) activity. Disruption of chu_1107 encoding ChCel5A caused no drastic effect on the growth parameters on cellulose for the resulting mutant strain with negligible reduction in the specific CMCase activities for intact cells. The demonstration of targeted gene inactivation capability for C. hutchinsonii has provided an opportunity to improve understanding of the details of the mechanism underlying its efficient utilization of cellulose.  相似文献   

14.
Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Targets were those amino acids identified to be important by 3D structure analysis of BsXynA in complex with substrate bound in the glycon subsites and the + 1 aglycon subsite. Several aromatic residues in the aglycon subsites that make strong substrate–protein interactions and that are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the + 2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that are involved in the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the + 3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the + 3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord – a long loop flanking the aglycon subsites of GH11 xylanases – proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.  相似文献   

15.
The glycoside hydrolase family 57 (GH57) contains five well-established enzyme specificities: α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and α-galactosidase. Around 700 GH57 members originate from Bacteria and Archaea, a substantial number being produced by thermophiles. An intriguing feature of family GH57 is that only slightly more than 2 % of its members (i.e., less than 20 enzymes) have already been biochemically characterized. The main goal of the present bioinformatics study was to retrieve from databases, and analyze in detail, sequences having clear features of the five GH57 enzyme specificities mentioned above. Of the 367 GH57 sequences, 56 were evaluated as α-amylases, 99 as amylopullulanases, 158 as branching enzymes, 46 as 4-α-glucanotransferases and 8 as α-galactosidases. Based on the analysis of collected sequences, sequence logos were created for each specificity and unique sequence features were identified within the logos. These features were proposed to define the so-called sequence fingerprints of GH57 enzyme specificities. Domain arrangements characteristic of the individual enzyme specificities as well as evolutionary relationships within the family GH57 are also discussed. The results of this study could find use in rational protein design of family GH57 amylolytic enzymes and also in the possibility of assigning a GH57 specificity to a hypothetical GH57 member prior to its biochemical characterization.  相似文献   

16.
BackgroundEnvironmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes.MethodsIn the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized.ResultsA phylogenetic analysis of the catalytic domain of pro- and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome.ConclusionsThe characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity.General significanceThe identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction.  相似文献   

17.
The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-β-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.  相似文献   

18.
In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat/K M) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.  相似文献   

19.
The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 A resolution. The presented structure model includes the catalytic and starch binding domains and traces the course of the 37-residue linker segment. While the structures of other fungal and yeast glucoamylase catalytic and starch binding domains have been determined separately, this is the first intact structure that allows visualization of the juxtaposition of the starch binding domain relative to the catalytic domain. The detailed interactions we see between the catalytic and starch binding domains are confirmed in a second independent structure determination of the enzyme in a second crystal form. This second structure model exhibits an identical conformation compared to the first structure model, which suggests that the H. jecorina glucoamylase structure we report is independent of crystal lattice contact restraints and represents the three-dimensional structure found in solution. The proposed starch binding regions for the starch binding domain are aligned with the catalytic domain in the three-dimensional structure in a manner that supports the hypothesis that the starch binding domain serves to target the glucoamylase at sites where the starch granular matrix is disrupted and where the enzyme might most effectively function.  相似文献   

20.
Wang G  Luo H  Meng K  Wang Y  Huang H  Shi P  Pan X  Yang P  Diao Q  Zhang H  Yao B 《PloS one》2011,6(2):e16731

Background

The rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.

Methodology/Principal Findings

We explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.

Conclusion/Significance

The numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.  相似文献   

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