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.

Scytalidium candidum 3C is a new name for the Geotrichum candidum Link 3C strain.

In the 1970s, the strain Geotrichum candidum Link 3C was isolated from rotting rope and since then has been extensively studied as a source of cellulose and xylan-degrading enzymes. The original identification of the strain was based only on morphological characters of the fungal mycelium in culture. Recent comparison of the internal transcribed spacer (ITS) fragments derived from the draft genome published in 2015 did not show its similarity to G. candidum species. Given the value of the strain 3C in lignocellulosic biomass degradation, we performed morphological and molecular studies to find the appropriate taxonomic placement for this fungal strain within the Ascomycota phylum. ITS, 18S rDNA, 28S rDNA sequences, and RPB2 encoding genes were used to construct phylogenetic trees with Maximum likelihood and Bayesian inference methods. Based on sequence comparison and multiple gene sequencing, we conclude that the fungal strain designated as Geotrichum candidum Link 3C should be placed into the genus Scytalidium (Pezizomycotina, Leotiomycetes) and is redescribed herein as Scytalidium candidum 3C comb. nov.

The novel strain Fusarium proliferatum LE1 (RCAM02409) produces α-L-fucosidase and arylsulfatase during the growth on fucoidan.

Enzymes capable of modifying the sulfated polymeric molecule of fucoidan are mainly produced by different groups of marine organisms: invertebrates, bacteria, and also some fungi. We have discovered and identified a new strain of filamentous fungus Fusarium proliferatum LE1 (deposition number in Russian Collection of Agricultural Microorganisms is RCAM02409), which is a potential producer of fucoidan-degrading enzymes. The strain LE1 (RCAM02409) was identified on the basis of morphological characteristics and analysis of ITS sequences of ribosomal DNA. During submerged cultivation of F. proliferatum LE1 in the nutrient medium containing natural fucoidan sources (the mixture of brown algae Laminaria digitata and Fucus vesiculosus), enzymic activities of α-L-fucosidase and arylsulfatase were inducible. These enzymes hydrolyzed model substrates, para-nitrophenyl α-L-fucopyranoside and para-nitrophenyl sulfate, respectively. However, the α-L-fucosidase is appeared to be a secreted enzyme while the arylsulfatase was an intracellular one. No detectable fucoidanase activity was found during F. proliferatum LE1 growth in submerged culture or in a static one. Comparative screening for fucoidanase/arylsulfatase/α-L-fucosidase activities among several related Fusarium strains showed a uniqueness of F. proliferatum LE1 to produce arylsulfatase and α-L-fucosidase enzymes. Apart them, the strain was shown to produce other glycoside hydrolyses.

Calcifying Bacteria Flexibility in Induction of CaCO3 Mineralization.

Microbially induced CaCO3 precipitation (MICP) is considered as an alternative green technology for cement self-healing and a basis for the development of new biomaterials. However, some issues about the role of bacteria in the induction of biogenic CaCO3 crystal nucleation, growth and aggregation are still debatable. Our aims were to screen for ureolytic calcifying microorganisms and analyze their MICP abilities during their growth in urea-supplemented and urea-deficient media. Nine candidates showed a high level of urease specific activity, and a sharp increase in the urea-containing medium pH resulted in efficient CaCO3 biomineralization. In the urea-deficient medium, all ureolytic bacteria also induced CaCO3 precipitation although at lower pH values. Five strains (B. licheniformis DSMZ 8782, B. cereus 4b, S. epidermidis 4a, M. luteus BS52, M. luteus 6) were found to completely repair micro-cracks in the cement samples. Detailed studies of the most promising strain B. licheniformis DSMZ 8782 revealed a slower rate of the polymorph transformation in the urea-deficient medium than in urea-containing one. We suppose that a ureolytic microorganism retains its ability to induce CaCO3 biomineralization regardless the origin of carbonate ions in a cell environment by switching between mechanisms of urea-degradation and metabolism of calcium organic salts.

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.

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.

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.

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.

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.

Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.

Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.