Tracing determinants of dual substrate specificity in glycoside hydrolase family 5

Enzymes are traditionally viewed as having exquisite substrate specificity; however, recent evidence supports the notion that many enzymes have evolved activities against a range of substrates. The diversity of activities across glycoside hydrolase family 5 (GH5) suggests that this family of enzymes may contain numerous members with activities on multiple substrates. In this study, we combined structure- and sequence-based phylogenetic analysis with biochemical characterization to survey the prevalence of dual specificity for glucan- and mannan-based substrates in the GH5 family. Examination of amino acid profile differences between the subfamilies led to the identification and subsequent experimental confirmation of an active site motif indicative of dual specificity. The motif enabled us to successfully discover several new dually specific members of GH5, and this pattern is present in over 70 other enzymes, strongly suggesting that dual endoglucanase-mannanase activity is widespread in this family. In addition, reinstatement of the conserved motif in a wild type member of GH5 enhanced its catalytic efficiency on glucan and mannan substrates by 175 and 1,600%, respectively. Phylogenetic examination of other GH families further indicates that the prevalence of enzyme multispecificity in GHs may be greater than has been experimentally characterized. Single domain multispecific GHs may be exploited for developing improved enzyme cocktails or facile engineering of microbial hosts for consolidated bioprocessing of lignocellulose.     

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)–linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.     

Degradative GH5 β-1,3-1,4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1

A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.     

Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (potential) commercial applications, e.g. in food, health and as biomaterials. Originally, the GH70 family was established only for glucansucrase enzymes of lactic acid bacteria that catalyze the synthesis of α-glucan polymers from sucrose. In recent years, we have identified 3 novel subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD), inactive on sucrose but converting starch/maltodextrin substrates into novel α-glucans. These novel starch-acting enzymes considerably enlarge the panel of α-glucans that can be produced. They also represent very interesting evolutionary intermediates between sucrose-acting GH70 glucansucrases and starch-acting GH13 α-amylases. Here we provide an overview of the repertoire of GH70 enzymes currently available with focus on these novel starch-acting GH70 enzymes and their biotechnological potential. Moreover, we discuss key developments in the understanding of structure-function relationships of GH70 enzymes in the light of available three-dimensional structures, and the protein engineering strategies that were recently applied to expand their natural product specificities.     

Enhancement of Synthetic Trichoderma-Based Enzyme Mixtures for Biomass Conversion with an Alternative Family 5 Glycosyl Hydrolase from Sporotrichum thermophile

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.     

Isozymes from the herbivorous gecarcinid land crab, Gecarcoidea natalis that possess both lichenase and endo-β-1,4-glucanase activity

Three isozymes with both lichenase and endo-β-1,4-glucanase activity were purified and characterised from the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. The three isozymes, termed 1a, 1b and 2, had respective molecular masses of 53 ± 0 (3), 43 ± 0 (3) and 47.4 ± 0(3) kDa. All isozymes possessed similar V(max) values and thus hydrolysed both carboxy methyl cellulose and lichenan equally. Furthermore the chromatography profiles for lichenase activities mirrored that for endo-β-1,4-glucanase activities suggesting that the same enzyme possessed both activities. Given this, the endo-β-1,4-glucanase enzymes described for other animals, may, like the isozymes described in this study, may be able to hydrolyse lichenan. However this ability needs to be confirmed. The main digestive function of these isozymes may be to hydrolyse hemicelluloses such as lichenan and mixed beta-D-glucan. All three isozymes randomly hydrolysed internal glycosidic bonds within carboxy methyl cellulose and lichenan to release short oligomers of 4-5 glucose units in length. They also hydrolysed cellotetraose to either two units of cellobiose or cellotriose and glucose. Cellotriose was hydrolysed to cellobiose and glucose. All three enzymes lacked β-1,4-glucosidase activity as they could not hydrolyse cellobiose.     

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.     

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

Background

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.

Results

CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.

Conclusion

We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

     

Phylogenomic Relationships between Amylolytic Enzymes from 85 Strains of Fungi

Fungal amylolytic enzymes, including α-amylase, gluocoamylase and α-glucosidase, have been extensively exploited in diverse industrial applications such as high fructose syrup production, paper making, food processing and ethanol production. In this paper, amylolytic genes of 85 strains of fungi from the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota were annotated on the genomic scale according to the classification of glycoside hydrolase (GH) from the Carbohydrate-Active enZymes (CAZy) Database. Comparisons of gene abundance in the fungi suggested that the repertoire of amylolytic genes adapted to their respective lifestyles. Amylolytic enzymes in family GH13 were divided into four distinct clades identified as heterologous α- amylases, eukaryotic α-amylases, bacterial and fungal α-amylases and GH13 α-glucosidases. Family GH15 had two branches, one for gluocoamylases, and the other with currently unknown function. GH31 α-glucosidases showed diverse branches consisting of neutral α-glucosidases, lysosomal acid α-glucosidases and a new clade phylogenetically related to the bacterial counterparts. Distribution of starch-binding domains in above fungal amylolytic enzymes was related to the enzyme source and phylogeny. Finally, likely scenarios for the evolution of amylolytic enzymes in fungi based on phylogenetic analyses were proposed. Our results provide new insights into evolutionary relationships among subgroups of fungal amylolytic enzymes and fungal evolutionary adaptation to ecological conditions.     

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Dividing the Large Glycoside Hydrolase Family 43 into Subfamilies: a Motivation for Detailed Enzyme Characterization

The rapid rise in DNA sequencing has led to an expansion in the number of glycoside hydrolase (GH) families. The GH43 family currently contains α-l-arabinofuranosidase, β-d-xylosidase, α-l-arabinanase, and β-d-galactosidase enzymes for the debranching and degradation of hemicellulose and pectin polymers. Many studies have revealed finer details about members of GH43 that necessitate the division of GH43 into subfamilies, as was done previously for the GH5 and GH13 families. The work presented here is a robust subfamily classification that assigns over 91% of all complete GH43 domains into 37 subfamilies that correlate with conserved sequence residues and results of biochemical assays and structural studies. Furthermore, cooccurrence analysis of these subfamilies and other functional modules revealed strong associations between some GH43 subfamilies and CBM6 and CBM13 domains. Cooccurrence analysis also revealed the presence of proteins containing up to three GH43 domains and belonging to different subfamilies, suggesting significant functional differences for each subfamily. Overall, the subfamily analysis suggests that the GH43 enzymes probably display a hitherto underestimated variety of subtle specificity features that are not apparent when the enzymes are assayed with simple synthetic substrates, such as pNP-glycosides.     

Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties

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.

     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.     

Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

A large polypeptide encoded in the genome of the thermophilic bacterium Caldicellulosiruptor bescii was determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol. 78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases from C. bescii led to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization by C. bescii.     

Properties of four C-terminal carbohydrate-binding modules (CBM4) of laminarinase Lic16A of Clostridium thermocellum

At the C-terminus of multimodular laminarinase Lic16A from Clostridium thermocellum, four carbohydrate-binding modules (CBM) of family 4 were found. Isolated CBM4_1, CBM4_2, CBM4_3, and CBM4_4 modules and the CBM4_(1-4) tandem were obtained. None of the recombinant proteins had the affinity to soluble ??-1,3-1,4-glucans, laminarin and lichenan, the main specific Lic16A substrates. All modules, except CBM4_4, had the ability to bind bacterial crystalline cellulose, which is atypical of family-4 CBMs. All CBMs 4 of Lic16A had an affinity to xylan, chitin, yeast cell wall ??-glucan, and avicel, while CBM4_3 and CBM4_4 also had an affinity to chitosan. The CBM4_(1-4) tandem had the highest affinity to the ??-glucan, avicel, and pustulan of the yeast cell wall. The CBM4_(1-4) binding constants for these substrates were approximately 100-fold higher than those of its individual modules, which suggests synergy in the process of absorbing these polysaccharides. This finding helps to explain the evolutionary process of CBM multiplication.     

Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis

This work describes the purification and characterization of enzymes that exhibit beta-d-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with beta-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative alpha-L-arabinofuranosidase, and XYL4 and XYL1 as putative beta-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-alpha-L-arabinofuranoside and XYL4 for p-nitrophenyl-beta-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a beta-D-xylosidase, while ARAf and XYL1 act as bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth.     

How family 26 glycoside hydrolases orchestrate catalysis on different polysaccharides: structure and activity of a Clostridium thermocellum lichenase, CtLic26A

One of the most intriguing features of the 90 glycoside hydrolase families (GHs) is the range of specificities displayed by different members of the same family, whereas the catalytic apparatus and mechanism are often invariant. Family GH26 predominantly comprises beta-1,4 mannanases; however, a bifunctional Clostridium thermocellum GH26 member (hereafter CtLic26A) displays a markedly different specificity. We show that CtLic26A is a lichenase, specific for mixed (Glcbeta1,4Glcbeta1,4Glcbeta1,3)n oligo- and polysaccharides, and displays no activity on manno-configured substrates or beta-1,4-linked homopolymers of glucose or xylose. The three-dimensional structure of the native form of CtLic26A has been solved at 1.50-A resolution, revealing a characteristic (beta/alpha)8 barrel with Glu-109 and Glu-222 acting as the catalytic acid/base and nucleophile in a double-displacement mechanism. The complex with the competitive inhibitor, Glc-beta-1,3-isofagomine (Ki 1 microm), at 1.60 A sheds light on substrate recognition in the -2 and -1 subsites and illuminates why the enzyme is specific for lichenan-based substrates. Hydrolysis of beta-mannosides by GH26 members is thought to proceed through transition states in the B2,5 (boat) conformation in which structural distinction of glucosides versus mannosides reflects not the configuration at C2 but the recognition of the pseudoaxial O3 of the B2,5 conformation. We suggest a different conformational itinerary for the GH26 enzymes active on gluco-configured substrates.     

Characterization of an endoglucanase belonging to a new subfamily of glycoside hydrolase family 45 of the basidiomycete Phanerochaete chrysosporium

The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley beta-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.