Xanthan: enzymatic degradation and novel perspectives of applications.

The extracellular heteropolysaccharide xanthan, synthesized by bacteria of the genus Xanthomonas, is widely used as a thickening and stabilizing agent across the food, cosmetic, and pharmaceutical sectors. Expanding the scope of its application, current efforts target the use of xanthan to develop innovative functional materials and products, such as edible films, eco-friendly oil surfactants, and biocompatible composites for tissue engineering. Xanthan-derived oligosaccharides are useful as nutritional supplements and plant defense elicitors. Development and processing of such new functional materials and products often necessitate tuning of xanthan properties through targeted structural modification. This task can be effectively carried out with the help of xanthan-specific enzymes. However, the complex molecular structure and intricate conformational behavior of xanthan create problems with its enzymatic hydrolysis or modification. This review summarizes and analyzes data concerning xanthan-degrading enzymes originating from microorganisms and microbial consortia, with a particular focus on the dependence of enzymatic activity on the structure and conformation of xanthan. Through a comparative study of xanthan-degrading pathways found within various bacterial classes, different microbial enzyme systems for xanthan utilization have been identified. The characterization of these new enzymes opens new perspectives for modifying xanthan structure and developing innovative xanthan-based applications. KEY POINTS: • The structure and conformation of xanthan affect enzymatic degradation. • Microorganisms use diverse multienzyme systems for xanthan degradation. • Xanthan-specific enzymes can be used to develop xanthan variants for novel applications.

Biochemical and Structural Characterization of Thermostable GH159 Glycoside Hydrolases Exhibiting α-L-Arabinofuranosidase Activity.

Functional, biochemical, and preliminary structural properties are reported for three glycoside hydrolases of the recently described glycoside hydrolase (GH) family 159. The genes were cloned from the genomic sequences of different Caldicellulosiruptor strains. This study extends the spectrum of functions of GH159 enzymes. The only activity previously reported for GH159 was hydrolytic activity on ß-galactofuranosides. Activity screening using a set of para-nitrophenyl (pNP) glycosides suggested additional arabinosidase activity on substrates with arabinosyl residues, which has not been previously reported for members of GH159. Even though the thermophilic enzymes investigated-Cs_Gaf159A, Ch_Gaf159A, and Ck_Gaf159A-cleaved pNP-α-l-arabinofuranoside, they were only weakly active on arabinogalactan, and they did not cleave arabinose from arabinan, arabinoxylan, or gum arabic. However, the enzymes were able to hydrolyze the α-1,3-linkage in different arabinoxylan-derived oligosaccharides (AXOS) with arabinosylated xylose at the non-reducing end (A3X, A2,3XX), suggesting their role in the intracellular hydrolysis of oligosaccharides. Crystallization and structural analysis of the apo form of one of the Caldicellulosiruptor enzymes, Ch_Gaf159A, enabled the elucidation of the first 3D structure of a GH159 member. This work revealed a five-bladed ß-propeller structure for GH159 enzymes. The 3D structure and its substrate-binding pocket also provides an explanation at the molecular level for the observed exo-activity of the enzyme. Furthermore, the structural data enabled the prediction of the catalytic amino acids. This was supported by the complete inactivation by mutation of residues D19, D142, and E190 of Ch_Gaf159A.

Activation of Subcutaneous Mast Cells in Acupuncture Points Triggers Analgesia.

This review summarizes experimental evidence indicating that subcutaneous mast cells are involved in the trigger mechanism of analgesia induced by acupuncture, a traditional oriental therapy, which has gradually become accepted worldwide. The results are essentially based on work from our laboratories. Skin mast cells are present at a high density in acupuncture points where fine needles are inserted and manipulated during acupuncture intervention. Mast cells are sensitive to mechanical stimulation because they express multiple types of mechanosensitive channels, including TRPV1, TRPV2, TRPV4, receptors and chloride channels. Acupuncture manipulation generates force and torque that indirectly activate the mast cells via the collagen network. Subsequently, various mediators, for example, histamine, serotonin, adenosine triphosphate and adenosine, are released from activated mast cells to the interstitial space; they or their downstream products activate the corresponding receptors situated at local nerve terminals of sensory neurons in peripheral ganglia. The analgesic effects are thought to be generated via the reduced electrical activities of the primary sensory neurons. Alternatively, these neurons project such signals to pain-relevant regions in spinal cord and/or higher centers of the brain.

Unusual substrate specificity in GH family 12: structure-function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris.

In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear ß-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris, purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides and had transglycosylase activity on (1,3)-ß-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a ß-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. KEY POINTS: • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides.

Strategic aromatic residues in the catalytic cleft of the xyloglucanase MtXgh74 modifying thermostability, mode of enzyme action, and viscosity reduction ability.

The thermostable endo-processive xyloglucanase MtXgh74 from Myceliophthora thermophila was used to study the influence of aromatic amino acids in the catalytic cleft on the mode of action and the ability of enzyme to reduce xyloglucan viscosity. The enzyme derivative Mut I with mutations W64A/W67A in the "negative" subsites of the catalytic cleft resulted in a 5.5-fold increase of the Km value. Mut I produced oligosaccharides of various lengths in addition to xyloglucan building blocks. The W320A/W321A substitutions in the "positive" subsites of the mutated enzyme Mut II catalytic cleft increased the Km value 54-fold and resulted in an endo-dissociative mode of action. The ability of Mut II to reduce the viscosity of xyloglucan at 50 °C was much better than that of other MtXgh74 variants. Besides, Mut II efficiently reduced viscosity of a natural substrate, the pulp of xyloglucan-containing tamarind seed flour. The Km, Vmax, and kcat values and viscosity reduction ability of the enzyme derivative Mut III (W320A/W321A/G446Y) returned to levels close to that of MtXgh74. The pattern of xyloglucan hydrolysis by Mut III was typical for endo-processive xyloglucanases. The thermostability of Mut I and Mut II at 60 °C decreased significantly compared to the wild type, whereas the thermostability of Mut III at 60 °C restored almost to the MtXgh74-wt value. All mutants lost the ability to cleave the backbone of xyloglucan building blocks which was a characteristic of MtXgh74. Instead they acquired a low branch removing activity. Molecular dynamics simulations revealed the role of mutated amino acids in the complex action mechanism of GH74 enzymes. KEY POINTS: • Endo-processive mode of action of the xyloglucanase MtXgh74 was altered by rational design. • The endo-dissociative mutant Mut II (W320A/W321A) efficiently reduced XyG viscosity. • The substitutions W320A/W321A/G446Y in Mut III recovered the endo-processive mode. • Mut II can be used to reduce the viscosity of biomass slurries containing tamarind seed flour.

Evaluation of the in vitro Function of Platelet Concentrates from Pooled Buffy Coats or Apheresis.

BACKGROUND: Platelet concentrates play an important role in transfusion medicine. Their short lifespan and lack of robustness require efforts to ensure adequate product quality. In this study, we compared the in vitro quality of the main concentrate types, pooled platelet concentrate (PPC) from whole blood donations, and platelet concentrate from single-donor apheresis (APC). METHODS: Twenty PPCs and 20 APCs prepared in plasma were analyzed on days 2, 4, and 7 of storage. Variables related to metabolism, degranulation, platelet aggregation, P-selectin expression, and annexin V binding were analyzed. Morphology was assessed by transmission electron microscopy of ultrathin sections. A microfluidic device was applied to test the effects of shear stress on platelet function. RESULTS: The metabolic parameters indicated stable storage conditions throughout the 7-day period. The resting discoid form was the prevailing morphology on days 2 and 4 in the PPCs and APCs. Chemokine release and receptor shedding of soluble P-selectin and soluble CD40L equally increased in PPCs and APCs. Aggregation responses to ADP and collagen were heterogeneous, with marked losses in collagen responsiveness on day 4 in individual concentrates. Baseline expression of P-selectin in PPCs and APCs was low, and inducibility of P-selectin was well preserved until day 4. Under shear stress, equal adhesiveness and stability were found with platelets from PPCs and APCs. CONCLUSIONS: Platelets from PPCs and APCs showed similar in vitro function and stability parameters. However, platelet concentrates presented a high variability and individual concentrates an impaired functional capability. Identifying the factors contributing to this would help increase product reliability.

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation.

Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful.Key points• Wheat milling byproducts are suitable substrates for clostridial ABE fermentation.• Enzymatic pretreatment of wheat red dog and middlings increases ABE yield.• ABE fermentation plants using wheat red dog as substrate are economically viable. Graphical abstract.

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containing GH48 gene sequences directly linked to the corresponding taxonomic information. Phylogenetic correlation analysis of GH48 to 16S rRNA gene sequences revealed that GH48 gene sequences with 94% identity belong with high confidence to the same genus. Applying this analysis, GH48 amplicon reads revealed that at mesophilic fermenter conditions, 50-99% of the OTUs appear to belong to novel taxa. In contrast, at thermophilic conditions, GH48 gene sequences from the genus Hungateiclostridium dominated with 60-91% relative abundance. The novel primer combinations enabled detection and relative quantification of a wide spectrum of GH48 genes in cellulolytic microbial communities. Deep phylogenetic correlation analysis and a simplified taxonomic identification with the novel database facilitate identification of cellulolytic organisms, including the detection of novel taxa in biogas fermenters.

P2Y13 and P2X7 receptors modulate mechanically induced adenosine triphosphate release from mast cells.

Subcutaneous mast cells (MCs) are vulnerable to mechanical stimulation from external environment. Thus, MCs immune function could be modulated by their mechanosensitivity. This property has been identified as the trigger mechanism of needling acupuncture, a traditional oriental therapy. Previously we have demonstrated the release of adenosine triphosphate (ATP), a stress-responsive signalling molecule, from mechanical-perturbed MCs. The current work explores its underlying mechanisms. We noticed that propagation of intracellular free Ca2+ occurred among HMC-1 cells in response to 50% hypotonic shock. Additionally, amplifying cascade of ATP-induced ATP release was observed in RBL-2H3 cells stimulated by medium displacement, which could be mimicked by exogenous ATP (exoATP). Secondary ATP liberation induced by low level (50 nmol/L) of exoATP was reduced by inhibiting ecto-ATPase-dependent ADP production with ARL67156, or blocking P2 receptors with suramin or PPADS, or with specific P2Y13 receptor antagonist MRS2211, or siRNA. Secondary ATP release induced by higher dose (200 µmol/L) of exoATP, sufficient to stimulate P2X7 receptor, was attenuated by suramin, PPADS or specific P2X7 receptor antagonist BBG, or siRNA. Finally, RT-PCR confirmed mRNA expression of P2Y13 and P2X7 in RBL-2H3 cells. Additionally, such secondary ATP release was attenuated by DPCPX, specific antagonist of adenosine A1 receptor, but not by MRS2179, specific inhibitor of P2Y1 receptor. In summary, mechanosensitive ATP release from MCs is facilitated by paracrine/autocrine stimulation of P2Y13 and P2X7 receptors. This multi-receptor combination could mediate transmission of information from a local site to distal areas, enabling communication with multiple surrounding cells to coordinate and synchronize their function.

Draft Genome Sequence of Paenibacillus polymyxa DSM 292, a Gram-Positive, Spore-Forming Soil Bacterium with High Biotechnological Potential.

Paenibacillus polymyxa DSM 292 was originally isolated from soil in 1947 due to its ability to produce antibiotics. The low proteolytic properties of strain DSM 292 warrant its examination as a host for heterologous protein production. Here, we report the draft genome sequence of DSM 292 as established by Illumina MiSeq paired-end sequencing.

Biochemical characterisation of four rhamnosidases from thermophilic bacteria of the genera Thermotoga, Caldicellulosiruptor and Thermoclostridium.

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm_Ram106B, Tn_Ram106B, Cb_Ram106B and Ts_Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-L-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as Km, Vmax and kcat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC50) between 5 mM and 8 mM. The α-L-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.

Novel endo-(1,4)-ß-glucanase Bgh12A and xyloglucanase Xgh12B from Aspergillus cervinus belong to GH12 subgroup I and II, respectively.

In spite of intensive exploitation of aspergilli for the industrial production of carbohydrases, little is known about hydrolytic enzymes of fungi from the section Cervini. Novel glycoside hydrolases Bgh12A and Xgh12B from Aspergillus cervinus represent examples of divergent activities within one enzyme family and belong to the GH12 phylogenetic subgroup I (endo-(1,4)-ß-glucanases) and II (endo-xyloglucanases), respectively. The bgh12A and xgh12B genes were identified in the unsequenced genome of A. cervinus using primers designed for conservative regions of the corresponding subgroups and a genome walking approach. The recombinant enzymes were heterologously produced in Pichia pastoris, purified, and characterized. Bgh12A was an endo-(1,4)-ß-glucanase (EC 3.2.1.4) hydrolyzing the unbranched soluble ß-(1,4)-glucans and mixed linkage ß-(1,3;1,4)-D-glucans. Bgh12A exhibited maximum activity on barley ß-glucan (BBG), which amounted to 614 ± 30 U/mg of protein. The final products of BBG and lichenan hydrolysis were glucose, cellobiose, cellotriose, 4-O-ß-laminaribiosyl-glucose, and a range of higher mixed-linkage gluco-oligosaccharides. In contrast, the activity of endo-xyloglucanase Xgh12B (EC 3.2.1.151) was restricted to xyloglucan, with 542 ± 39 U/mg protein. The enzyme cleaved the (1,4)-ß-glycosidic bonds of the xyloglucan backbone at the unsubstituted glucose residues finally generating cellotetraose-based hepta-, octa, and nona-oligosaccharides. Bgh12A and Xgh12B had maximal activity at 55 °C, pH 5.0. At these conditions, the half-time of Xgh12B inactivation was 158 min, whereas the half-life of Bgh12A was 5 min. Recombinant P. pastoris strains produced up to 106 U/L of the target enzymes with at least 75% of recombinant protein in the total extracellular proteins. The Bgh12A and Xgh12B sequences show 43% identity. Strict differences in substrate specificity of Bgh12A and Xgh12B were in congruence with the presence of subgroup-specific structural loops and substrate-binding aromatic residues in the catalytic cleft of the enzymes. Individual composition of aromatic residues in the catalytic cleft defined variability in substrate selectivity within GH12 subgroups I and II.

Addition of ß-galactosidase boosts the xyloglucan degradation capability of endoglucanase Cel9D from Clostridium thermocellum.

BACKGROUND: Increasing the efficiency of enzymatic biomass degradation is crucial for a more economically feasible conversion of abundantly available plant feedstock. Synergistic effects between the enzymes deployed in the hydrolysis of various hemicelluloses have been demonstrated, which can reduce process costs by lowering the amount of enzyme required for the reaction. Xyloglucan is the only major hemicellulose for which no such effects have been described yet. RESULTS: We report the beneficial combination of two enzymes for the degradation of the hemicellulose xyloglucan. The addition of ß-galactosidase Bga2B from Clostridium stercorarium to an in vitro hydrolysis reaction of a model xyloglucan substrate increased the enzymatic efficiency of endoglucanase Cel9D from Clostridium thermocellum to up to 22-fold. Furthermore, the total amount of enzyme required for high hydrolysis yields was lowered by nearly 80%. Increased yields were also observed when using a natural complex substrate-tamarind kernel powder. CONCLUSION: The findings of this study may improve the valorization of feedstocks containing high-xyloglucan amounts. The combination of the endoglucanase Cel9D and the ß-galactosidase Bga2B can be used to efficiently produce the heptasaccharide XXXG. The exploitation of one specific oligosaccharide may open up possibilities for the use as a prebiotic or platform chemical in additional reactions.

Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa.

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

Optimizing the composition of a synthetic cellulosome complex for the hydrolysis of softwood pulp: identification of the enzymatic core functions and biochemical complex characterization.

BACKGROUND: The development of efficient cellulase blends is a key factor for cost-effectively valorizing biomass in a new bio-economy. Today, the enzymatic hydrolysis of plant-derived polysaccharides is mainly accomplished with fungal cellulases, whereas potentially equally effective cellulose-degrading systems from bacteria have not been developed. Particularly, a thermostable multi-enzyme cellulase complex, the cellulosome from the anaerobic cellulolytic bacterium Clostridium thermocellum is promising of being applied as cellulolytic nano-machinery for the production of fermentable sugars from cellulosic biomass. RESULTS: In this study, 60 cellulosomal components were recombinantly produced in E. coli and systematically permuted in synthetic complexes to study the function-activity relationship of all available enzymes on Kraft pulp from pine wood as the substrate. Starting from a basic exo/endoglucanase complex, we were able to identify additional functional classes such as mannanase and xylanase for optimal activity on the substrate. Based on these results, we predicted a synthetic cellulosome complex consisting of seven single components (including the scaffoldin protein and a ß-glucosidase) and characterized it biochemically. We obtained a highly thermostable complex with optimal activity around 60-65 °C and an optimal pH in agreement with the optimum of the native cellulosome (pH 5.8). Remarkably, a fully synthetic complex containing 47 single cellulosomal components showed comparable activity with a commercially available fungal enzyme cocktail on the softwood pulp substrate. CONCLUSIONS: Our results show that synthetic bacterial multi-enzyme complexes based on the cellulosome of C. thermocellum can be applied as a versatile platform for the quick adaptation and efficient degradation of a substrate of interest.

The hemicellulose-degrading enzyme system of the thermophilic bacterium Clostridium stercorarium: comparative characterisation and addition of new hemicellulolytic glycoside hydrolases.

BACKGROUND: The bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. Clostridium stercorarium is a thermophilic bacterium, well known for its outstanding hemicellulose-degrading capability. Its genome comprises about 50 genes for partially still uncharacterised thermostable hemicellulolytic enzymes. These are promising candidates for industrial applications. RESULTS: To reveal the hemicellulose-degrading potential of 50 glycoside hydrolases, they were recombinantly produced and characterised. 46 of them were identified in the secretome of C. stercorarium cultivated on cellobiose. Xylanases Xyn11A, Xyn10B, Xyn10C, and cellulase Cel9Z were among the most abundant proteins. The secretome of C. stercorarium was active on xylan, ß-glucan, xyloglucan, galactan, and glucomannan. In addition, the recombinant enzymes hydrolysed arabinan, mannan, and galactomannan. 20 enzymes are newly described, degrading xylan, galactan, arabinan, mannan, and aryl-glycosides of ß-d-xylose, ß-d-glucose, ß-d-galactose, α-l-arabinofuranose, α-l-rhamnose, ß-d-glucuronic acid, and N-acetyl-ß-d-glucosamine. The activities of three enzymes with non-classified glycoside hydrolase (GH) family modules were determined. Xylanase Xyn105F and ß-d-xylosidase Bxl31D showed activities not described so far for their GH families. 11 of the 13 polysaccharide-degrading enzymes were most active at pH 5.0 to pH 6.5 and at temperatures of 57-76 °C. Investigation of the substrate and product specificity of arabinoxylan-degrading enzymes revealed that only the GH10 xylanases were able to degrade arabinoxylooligosaccharides. While Xyn10C was inhibited by α-(1,2)-arabinosylations, Xyn10D showed a degradation pattern different to Xyn10B and Xyn10C. Xyn11A released longer degradation products than Xyn10B. Both tested arabinose-releasing enzymes, Arf51B and Axh43A, were able to hydrolyse single- as well as double-arabinosylated xylooligosaccharides. CONCLUSIONS: The obtained results lead to a better understanding of the hemicellulose-degrading capacity of C. stercorarium and its involved enzyme systems. Despite similar average activities measured by depolymerisation tests, a closer look revealed distinctive differences in the activities and specificities within an enzyme class. This may lead to synergistic effects and influence the enzyme choice for biotechnological applications. The newly characterised glycoside hydrolases can now serve as components of an enzyme platform for industrial applications in order to reconstitute synthetic enzyme systems for complete and optimised degradation of defined polysaccharides and hemicellulose.

Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol.

BACKGROUND: The genus Bacillus includes a great variety of species with potential applications in biotechnology. While species such as B. subtilis or B. licheniformis are well-known and used to provide various products at industrial scale, other Bacillus species are less characterized and are not yet used in commercial processes. One reason for this is the fact that genetic manipulation of new isolates is usually complicated with conventional techniques which have to be adapted to each new strain. Even in well-established strains, the available transformation protocols often suffer from low efficiencies. RESULTS: In this paper, we provide a new broad host range E. coli/Bacillus shuttle vector, named pBACOV (Bacillus conjugation vector), that can be efficiently transferred to various Bacillus species using a single protocol. A variant of pBACOV carrying the sfGFP gene was successfully transferred to eight different species from the genus Bacillus and to one Paenibacillus species using triparental conjugation ("transmating"). This was achieved using a single protocol and worked for nine out of eleven tested acceptor species. The transmating procedure was used to test expression of the heterologous reporter gene sfGFP under control of the PaprE-promoter from B. subtilis in several Bacillus species in parallel. Expression of sfGFP was found in eight out of nine transmates. For several of the tested species, this is the first report of a method for genetic modification and heterologous gene expression. The expression level, analyzed by measuring the relative sfGFP-fluorescence normalized to the cell density of the cultures, was highest in B. mojavensis. CONCLUSIONS: The new shuttle vector pBACOV can be transferred to many different Bacillus and Paenibacillus species using a simple and efficient transmating protocol. It is a versatile tool facilitating the application of recombinant DNA technology in new as well as established strains, or selection of an ideal host for heterologous gene expression from a multitude of strains. This paves the way for the genetic modification and biotechnological exploitation of the broad diversity of species of Bacillus and related genera as well as different strains from these species.

Handling gene and protein names in the age of bioinformatics: the special challenge of secreted multimodular bacterial enzymes such as the cbhA/cbh9A gene of Clostridium thermocellum.

An increasing number of researchers working in biology, biochemistry, biotechnology, bioengineering, bioinformatics and other related fields of science are using biological molecules. As the scientific background of the members of different scientific communities is more diverse than ever before, the number of scientists not familiar with the rules for non-ambiguous designation of genetic elements is increasing. However, with biological molecules gaining importance through biotechnology, their functional and unambiguous designation is vital. Unfortunately, naming genes and proteins is not an easy task. In addition, the traditional concepts of bioinformatics are challenged with the appearance of proteins comprising different modules with a respective function in each module. This article highlights basic rules and novel solutions in designation recently used within the community of bacterial geneticists, and we discuss the present-day handling of gene and protein designations. As an example we will utilize a recent mischaracterization of gene nomenclature. We make suggestions for better handling of names in future literature as well as in databases and annotation projects. Our methodology emphasizes the hydrolytic function of multi-modular genes and extracellular proteins from bacteria.

Complete Genome Sequence of the Novel Cellulolytic, Anaerobic, Thermophilic Bacterium Herbivorax saccincola Type Strain GGR1, Isolated from a Lab Scale Biogas Reactor as Established by Illumina and Nanopore MinION Sequencing.

The cellulolytic bacterium Herbivorax saccincola strain GGR1, which represents the type strain of this species, was isolated from the in vivo enriched cellulose-binding community of a lab scale thermophilic biogas reactor. Here, we report the complete genome sequence of H. saccincola GGR1T, the first isolated member of the genus Herbivorax.

Сarbohydrate binding module CBM28 of endoglucanase Cel5D from Caldicellulosiruptor bescii recognizes crystalline cellulose.

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·104 and 2.2·106M-1 for cellotetraose and cellohexaose, respectively. The interaction between CbСВМ28 and amorphous cellulose is best described by a two-binding-site model with the binding constants of 1.5·105 and 1.9·105M-1. In a competitive binding assay in the presence of a 10-fold excess of cellohexaose the binding constant of CbСВМ28 to amorphous cellulose was 1.9·105M-1. A two-binding-site model also better approximates the binding to Avicel with the binding constants of 8.3·105 and 3.2·104M-1; while in the presence of cellohexaose, the binding is described by a single-binding-site model with the binding constant of 2.3·104M-1. With CbСВМ28 binding to bacterial crystalline cellulose with a constant of 7.4·104M-1, this is the first report of such a strong binding to crystalline cellulose for a module of family 28.