Forgotten Actors: Glycoside Hydrolases During Elongation Growth of Maize Primary Root.

Plant cell enlargement is coupled to dynamic changes in cell wall composition and properties. Such rearrangements are provided, besides the differential synthesis of individual cell wall components, by enzymes that modify polysaccharides in muro. To reveal enzymes that may contribute to these modifications and relate them to stages of elongation growth in grasses, we carried out a transcriptomic study of five zones of the primary maize root. In the initiation of elongation, significant changes occur with xyloglucan: once synthesized in the meristem, it can be linked to other polysaccharides through the action of hetero-specific xyloglucan endotransglycosidases, whose expression boosts at this stage. Later, genes for xyloglucan hydrolases are upregulated. Two different sets of enzymes capable of modifying glucuronoarabinoxylans, mainly bifunctional α-arabinofuranosidases/ß-xylosidases and ß-xylanases, are expressed in the maize root to treat the xylans of primary and secondary cell walls, respectively. The first set is highly pronounced in the stage of active elongation, while the second is at elongation termination. Genes encoding several glycoside hydrolases that are able to degrade mixed-linkage glucan are downregulated specifically at the active elongation. It indicates the significance of mixed-linkage glucans for the cell elongation process. The possibility that many glycoside hydrolases act as transglycosylases in muro is discussed.

Nonspecific enzymatic hydrolysis of a highly ordered chitopolysaccharide substrate.

Chitin and chitosan can undergo nonspecific enzymatic hydrolysis by several different hydrolases. This susceptibility to nonspecific enzymes opens up many opportunities for producing chitooligosaccharides and low molecular weight chitopolysaccharides, since specific chitinases and chitosanases are rare and not commercially available. In this study, chitosan and chitin were hydrolyzed using several commercially available hydrolases. Among them, cellulases with the highest specific activity demonstrated the best activity, as indicated by the rapid decrease in viscosity of a chitosan solution. The hydrolysis of chitosan by nonspecific enzymes generated a sugar release that corresponded to the decrease in the degree of polymerization. This decrease reached a maximum of 3.3-fold upon hydrolysis of 10% of the sample. Cellulases were better than lysozyme or amylases at hydrolyzing chitosan and chitin. Analysis of 13C CP-MAS NMR and FTIR spectra of chitin after cellulase treatment revealed changes in the chitin crystal structure related to rearrangement of inter- and intramolecular H-bonds. The structural changes and decreases in crystallinity allowed dissolution of chitin molecules of high molecular weight and enhanced the solubility of chitin in alkali by 10-12% compared to untreated chitin.

A novel acid-tolerant ß-xylanase from Scytalidium candidum 3C for the synthesis of o-nitrophenyl xylooligosaccharides.

Endo-ß-xylanases are hemicellulases involved in the conversion of xylans in plant biomass. Here, we report a novel acidophilic ß-xylanase (ScXynA) with high transglycosylation abilities that was isolated from the filamentous fungus Scytalidium candidum 3C. ScXynA was identified as a glycoside hydrolase family 10 (GH10) dimeric protein, with a molecular weight of 38 ± 5 kDa per subunit. The enzyme catalyzed the hydrolysis of different xylans under acidic conditions and was stable in the pH range 2.6-4.5. The kinetic parameters of ScXynA were determined in hydrolysis reactions with p-nitrophenyl-ß-d-cellobioside (pNP-ß-Cel) and p-nitrophenyl-ß-d-xylobioside (pNP-ß-Xyl2 ), and kcat /Km was found to be 0.43 ± 0.02 (s·mM)-1 and 57 ± 3 (s·mM)-1 , respectively. In the catalysis of the transglycosylation o-nitrophenyl-ß-d-xylobioside (oNP-ß-Xyl2 ) acted both as a donor and an acceptor, resulting in the efficient production of o-nitrophenyl xylooligosaccharides, with a degree of polymerization of 3-10 and o-nitrophenyl-ß-d-xylotetraose (oNP-ß-Xyl4 ) as the major product (18.5% yield). The modeled ScXynA structure showed a favorable position for ligand entry and o-nitrophenyl group accommodation in the relatively open -3 subsite, while the cleavage site was covered with an extended loop. These structural features provide favorable conditions for transglycosylation with oNP-ß-Xyl2 . The acidophilic properties and high transglycosylation activity make ScXynA a suitable choice for various biotechnological applications, including the synthesis of valuable xylooligosaccharides.

Crystal and Supramolecular Structure of Bacterial Cellulose Hydrolyzed by Cellobiohydrolase from Scytalidium Candidum 3C: A Basis for Development of Biodegradable Wound Dressings.

The crystal and supramolecular structure of the bacterial cellulose (BC) has been studied at different stages of cellobiohydrolase hydrolysis using various physical and microscopic methods. Enzymatic hydrolysis significantly affected the crystal and supramolecular structure of native BC, in which the 3D polymer network consisted of nanoribbons with a thickness T ≈ 8 nm and a width W ≈ 50 nm, and with a developed specific surface SBET ≈ 260 m2·g-1. Biodegradation for 24 h led to a ten percent decrease in the mean crystal size Dhkl of BC, to two-fold increase in the sizes of nanoribbons, and in the specific surface area SBET up to ≈ 100 m2·g-1. Atomic force and scanning electron microscopy images showed BC microstructure "loosening"after enzymatic treatment, as well as the formation and accumulation of submicron particles in the cells of the 3D polymer network. Experiments in vitro and in vivo did not reveal cytotoxic effect by the enzyme addition to BC dressings and showed a generally positive influence on the treatment of extensive III-degree burns, significantly accelerating wound healing in rats. Thus, in our opinion, the results obtained can serve as a basis for further development of effective biodegradable dressings for wound healing.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum.

BACKGROUND: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. RESULTS: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. CONCLUSIONS: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.

Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1.

Sulfatases are a family of enzymes (sulfuric ester hydrolases, EC 3.1.6.-) that catalyze the hydrolysis of a wide array of sulfate esters. To date, despite the discovery of many sulfatase genes and the accumulation of data on numerous sulfated molecules, the number of characterized enzymes that are key players in sulfur metabolism remains extremely limited. While mammalian sulfatases are well studied due to their involvement in a wide range of normal and pathological biological processes, lower eukaryotic sulfatases, especially fungal sulfatases, have not been thoroughly investigated at the biochemical and structural level. In this paper, we describe the molecular cloning of Fusarium proliferatum sulfatase (F.p.Sulf-6His), its recombinant expression in Pichia pastoris as a soluble and active cytosolic enzyme and its detailed characterization. Gel filtration and native electrophoretic experiments showed that this recombinant enzyme exists as a tetramer in solution. The enzyme is thermo-sensitive, with an optimal temperature of 25°C. The optimal pH value for the hydrolysis of sulfate esters and stability of the enzyme was 6.0. Despite the absence of the post-translational modification of cysteine into Cα-formylglycine, the recombinant F.p.Sulf-6His has remarkably stable catalytic activity against p-nitrophenol sulfate, with kcat = 0.28 s-1 and Km = 2.45 mM, which indicates potential use in the desulfating processes. The currently proposed enzymatic mechanisms of sulfate ester hydrolysis do not explain the appearance of catalytic activity for the unmodified enzyme. According to the available models, the unmodified enzyme is not able to perform multiple catalytic acts; therefore, the enzymatic mechanism of sulfate esters hydrolysis remains to be fully elucidated.

Characterization of a new α-l-fucosidase isolated from Fusarium proliferatum LE1 that is regioselective to α-(1 → 4)-l-fucosidic linkage in the hydrolysis of α-l-fucobiosides.

Here, we report the biochemical characterization of a novel α-l-fucosidase with broad substrate specificity (FpFucA) isolated from the mycelial fungus Fusarium proliferatum LE1. Highly purified α-l-fucosidase was obtained from several chromatographic steps after growth in the presence of l-fucose. The purified α-l-fucosidase appeared to be a monomeric protein of 67 ± 1 kDa that was able to hydrolyze the synthetic substrate p-nitrophenyl α-l-fucopyranoside (pNPFuc), with Km = 1.1 ± 0.1 mM and kcat = 39.8 ± 1.8 s-1. l-fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited pNPFuc hydrolysis, with inhibition constants of 0.2 ± 0.05 mM, 7.1 ± 0.05 nM, and 12.2 ± 0.1 mM, respectively. We assumed that the enzyme belongs to subfamily A of the GH29 family (CAZy database) based on its ability to hydrolyze practically all fucose-containing oligosaccharides used in the study and the phylogenetic analysis. We found that this enzyme was a unique α-l-fucosidase that preferentially hydrolyzes the α-(1 â†’ 4)-L-fucosidic linkage present in α-L-fucobiosides with different types of linkages. As a retaining glycosidase, FpFucA is capable of catalyzing the transglycosylation reaction with alcohols (methanol, ethanol, and 1-propanol) and pNP-containing monosaccharides as acceptors. These features make the enzyme an important tool that can be used in the various modifications of valuable fucose-containing compounds.

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C.

The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60-65 °C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported--on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925. ENZYMES: Glycoside hydrolase family 7 reducing end acting cellobiohydrolase.

The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolysis of p-nitrophenyl glycosides.

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, ß-xylosidase from Aspergillus awamori, ß-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.

Xylan degradation improved by a combination of monolithic columns bearing immobilized recombinant ß-xylosidase from Aspergillus awamori X-100 and Grindamyl H121 ß-xylanase.

Synergistic action of exo- and endohydrolazes is preferred for effective destruction of biopolymers. The main purpose of the present work was to develop an efficient tool for degradation of xylan. Macroporous lab-made monolithic columns and commercial CIM-Epoxy disk were used to immobilize the recombinant ß-xylosidase from Aspergillus awamori and Grindamyl ß-xylanase. The efficiency of xylan degradation using the low-loaded ß-xylosidase column appeared to be four times higher than for the in-solution process and about six times higher than for the high-loaded bioreactor. Disk bioreactor with the Grindamil ß-xylanase operated in a recirculation mode has shown noticeable advantages over the column design. Additionally, a system comprised of two immobilized enzyme reactors (IMERs) was tested to accelerate the biopolymer hydrolysis, yielding total xylan conversion into xylose within 20 min. Fast online monitoring HPLC procedure was developed where an analytical DEAE CIM disk was added to the two-enzyme system in a conjoint mode. A loss of activity of immobilized enzymes did not exceed 7% after 5 months of the bioreactor usage. We can therefore conclude that the bioreactors developed exhibit high efficiency and remarkable long-term stability.

α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima.

Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG(‡)0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG(‡)0=1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG(‡)F-ΔΔG(‡)H), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower.

Draft Genome Sequence of Geotrichum candidum Strain 3C.

We report here the draft genome sequence of Geotrichum candidum strain 3C, which is a filamentous yeast-like fungus that holds great promise for biotechnology. The genome was sequenced using Ion Torrent and 454 platforms. The estimated genome size was 41.4 Mb, and 14,579 protein-coding genes were predicted ab initio.

Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus.

To understand structure-function relationships in the N-terminal region of GH11 xylanases, the 17 N-terminal amino acids of the GH11 xylanase from Neocallimastix patriciarum (Np-Xyn) have been grafted onto the N-terminal extremity of the untypically short GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn), creating a hybrid enzyme denoted NTfus. The hybrid xylanase displayed properties (pH and temperature optima) similar to those of the parental enzyme, although thermostability was lowered, with the Tm value, being reduced by 5°C. Kinetic assays using oNP-Xylo-oligosaccharides (DP2 and 3) indicated that the N-extension did not procure more extensive substrate binding, even when further mutagenesis was performed to promote this. However, these experiments confirmed weak subsite -3 for both NTfus and the parental enzyme. The catalytic efficiency of NTfus was shown to be 17% higher than that of the parental enzyme on low viscosity wheat arabinoxylan and trials using milled wheat straw as the substrate revealed that NTfus released more substituted oligosaccharide products (Xyl/Ara=8.97±0.13 compared to Xyl/Ara=9.70±0.21 for the parental enzyme), suggesting that the hybrid enzyme possesses wider substrate selectivity. Combining either the parental enzyme or NTfus with the cellulolytic cocktail Accellerase 1500 boosted the impact of the latter on wheat straw, procuring yields of solubilized xylose and glucose of 23 and 24% of theoretical yield, respectively, thus underlining the benefits of added xylanase activity when using this cellulase cocktail. Overall, in view of the results obtained for NTfus, we propose that the N-terminal extension leads to the modification of a putative secondary substrate binding site, a hypothesis that is highly consistent with previous data.

Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases.

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.

Transglycosylating and hydrolytic activities of the beta-mannosidase from Trichoderma reesei.

A purified beta-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl beta-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing beta-(1-->4) and beta-(1-->3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.

Crystallization and preliminary crystallographic analysis of laminarinase from Rhodothermus marinus: a case of pseudomerohedral twinning.

Thermophilic endo-1,3(4)-beta glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and contain two protein molecules in the asymmetric unit with a solvent content of 51.75 %. Diffraction data were collected to a resolution of 1.95A and resulted in a dataset with an overall R(merge) of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.