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Highlights • R. josui Abf43A-Abf43B-Abf43C contains 3 catalytic modules classified in family GH43. • Abf43A, Abf43B and Abf43C are classified in different subfamilies of GH43. • Abf43A is a branched arabinan-specific a- l -arabinofuranosidase. • Abf43B and Abf43C are exo-a-1,5- l -arabinofuranosidases. • Abf43A and Abf43B synergistically acted on sugar beet arabinan and sugar beet fiber. Abstract The abnA gene from Ruminiclostridium josui encodes the large modular arabinanolytic enzyme, Abf43A-Abf43B-Abf43C, consisting of an N-terminal signal peptide, a Laminin_G_3 module, a GH43_22 module, a Laminin_G_3 module, a Big_4 module, a GH43_26 module, a GH43_34 module and a dockerin module in order with a calculated molecular weight of 204,108. Three truncated enzymes were recombinantly produced in Escherichia coli and biochemically characterized, RjAbf43A consisting of the first Laminin_G_3 module and GH43_22 module, RjAbf43B consisting of the second Laminin_G_3 module, Big_4 module and GH43_26 module, and RjAbf43C consisting of the GH43_34 module. RjAbf43A showed a strong α- l -arabinofuranosidase activity toward sugar beet arabinan, highly branched arabinan but not linear arabinan, thus it acted in the removal of arabinose side chains from sugar beet arabinan. By contrast, RjAbf43B showed a strong exo-α-1,5- l -arabinofuranosidase activity toward linear arabinan and arabinooligosaccharides whereas RjAbf43C showed low activity toward these substrates. Although RjAbf43B was activated by the presence of some metal ions such as Zn2+, Mg2+ and Ni2+, RjAbf43A was inhibited by these ions. RjAbf43A and RjAbf43B attacked sugar beet arabinan in a synergistic manner. By comparison, RjAbf43A-Abf43B containing both GH43_22 and GH43_26 modules showed lower hydrolytic activity toward sugar beet arabinan but higher activity toward sugar beet fiber than the sum of the individual activities of RjAbf43A and RjAbf43B, suggesting that the coexistence of two distinct GH43 modules in a single polypeptide is important for the efficient hydrolysis of an insoluble and natural polysaccharide but not a soluble substrate. [ABSTRACT FROM AUTHOR]

Laminin_G_3 modules can exist together with family‐43 catalytic modules of glycoside hydrolase (GH43), but their functions are unknown. Here, a laminin_G_3 module and a GH43 module derived from a Ruminiclostridium josui modular arabinofuranosidase Abf43A‐Abf43B‐Abf43C were produced individually as RjLG3 and RjGH43_22, respectively, or combined as RjGH43‐1 to gain insights into their activities. Isothermal calorimetry analysis showed that RjLG3 has high affinity toward 32‐α‐l‐arabinofuranosyl‐(1,5)‐α‐l‐arabinotriose but not for α‐1,5‐linked arabinooligosaccharides, which suggests that RjLG3 interacts specifically with a branched arabinofuranosyl residue of an arabinooligosaccharide but not an arabinofuranosyl residue at the end of α‐1,5‐linked arabinooligosaccharides. RjGH43‐1 (with CBM) shows higher activity toward sugar beet arabinan than RjGH43_22 (without CBM), which suggests that the LG3 module in RjGH43‐1 plays an important role in substrate hydrolysis as a carbohydrate‐binding module. [ABSTRACT FROM AUTHOR]

Ruminiclostridium josui Abf62A-Axe6A is a modular enzyme comprising (in order from the N-terminus): an N-terminal signal peptide, a glycoside hydrolase family 62 (GH62) catalytic module, a family 6 carbohydrate binding module (CBM6), a dockerin module and an additional carbohydrate esterase family 6 catalytic module (CE6). In this study, three Abf62A-Axe6A derivatives were constructed, overexpressed in Escherichia coli, purified, and biochemically characterized: RjAbf62A-Axe6A, containing all four modules but lacking the signal peptide; RjAbf62A-CBM6, containing the GH62 and CBM6 modules; and RjAxe6A, containing only CE6. RjAbf62A-Axe6A was highly active toward arabinoxylan and moderately active toward sugar beet arabinan, and released mainly arabinose. Analysis of the arabinoxylooligosaccharide hydrolysis products revealed that RjAbf62A-Axe6A released α-1,2- and α-1,3-linked arabinofuranose from both singly and doubly substituted xylosyl residues. Furthermore, RjAbf62A-Axe6A exhibited a weak activity toward linear 1,5-α- l arabinan and arabinooligosaccharides, indicating that it is capable of cleaving α-1,5-linkage. Surprisingly, RjAbf62A-Axe6A also demonstrated an endoxylanase activity toward birchwood and beechwood xylans and xylooligosaccharides. Although RjAbf62A-CBM6 exhibited a similar substrate specificity to RjAbf62A-Axe6A, RjAbf62A-CBM6 showed lower activities toward soluble arabinoxylans, birchwood and beechwood xylans and arabinoxylooligosaccharides but not toward insoluble arabinoxylan. RjAbf62A-Axe6A is the first reported GH62 enzyme with α- l -arabinofuranosidase and endoxylanase activities. Although both RjAbf62A-Axe6A and RjAxe6A had acetylxylan esterase activities, RjAbf62A-Axe6 exhibited a higher activity toward insoluble wheat arabinoxylan compared with RjAxe6. [ABSTRACT FROM AUTHOR]

A novel gene ( axh43B ) from Ruminiclostridium josui encoding a cellulosomal enzyme consisting of a catalytic module of subfamily GH43_10, a family-6 carbohydrate-binding module, and a dockerin module, was expressed using Escherichia coli . RjAxh43 B released only arabinose from arabinoxylan and 2 3 ,3 3 -di-α- l -arabinofuranosyl xylotriose, but not 3 2 -α- l -arabinofuranosyl xylobiose or 2 3 -α- l -arabinofuranosyl xylotriose, strongly suggesting that RjAxh43 B is an arabinoxylan α- l -1,3-arabinofuranohydrolase capable of cleaving α-1,3-linked arabinose residues of doubly arabinosylated xylan. When Axh43 B was mixed with the recombinant scaffolding protein RjCipA of R. josui at a molar ratio of 6:1, the activity of the RjAxh43B-RjCipA complex (6:1) toward insoluble wheat arabinoxylan was similar to that of RjAxh43 B alone, suggesting that RjAxh43 B does not show a proximity effect, which is defined as an activity enhancement effect caused by the presence of plural catalytic subunits adjoining each other. When RjAxh43A was mixed with xylanase RjXyn10C, they acted synergistically toward insoluble wheat arabinoxylan and rice straw powder in the absence of RjCipA. Furthermore, the RjAxh43B-RjXyn10C-RjCipA (3:3:3) complex had higher activity toward insoluble wheat arabinoxylan than a mixture of RjAxh43 B and RjXyn10C without RjCipA, suggesting that incorporation of a xylanase and an α- l -arabinofuranosidase into a cellulosome is beneficial for more efficiently degrading arabinoxylan. [ABSTRACT FROM AUTHOR]

Bacillus safensis M3 was newly isolated from freshwater swamp forest soil in western Thailand. The endoglucanase gene of B. safensis M3, cel9A, had an open reading frame of 1,848 bp encoding a 616 amino acid protein. Initial expression in Escherichia coli yielded a low amount of soluble protein in the cytosolic and secreted fractions. Cel9A was successfully expressed by recombinant B. subtilis with a 4- fold greater total enzyme activity than from recombinant E. coli. By SDS-PAGE analysis, the molecular weight of Cel9A was estimated to be 70 kDa. The optimal temperature of Cel9A was 55 °C and the optimal pH was 5 - 8. Cel9A had the highest activity in the pH range from 5 - 8 and the highest stability in pH range 4 to 10, which is useful for industrial applications. Notably, Cel9A was able to hydrolyze both mixed linkage glucan (lichenan) and hemicellulose (konjac glucomannan and oat spelt xylan) better than carboxymethylcellulose. Cel9A also showed a tolerance to metal ions and surfactants. In addition, recombinant B. subtilis with endoglucanase activity has potential for biotechnological applications and benefits in the optimization of large scale enzyme production with minimal medium using agriculture wastes and other inexpensive feedstock materials. [ABSTRACT FROM AUTHOR]

Three cellulosomal subunits of Ruminiclostridium josui , the full-length scaffolding protein CipA (RjCipA), a cellulase Cel5B (RjCel5B) and a xylanase Xyn10C (RjXyn10C), were successfully produced by Escherichia coli recombinant clones. RjCel5B and RjXyn10C were characterized as an endoglucanase and an endoxylanase, respectively. RjCipA, RjCel5B and Xyn10C adsorbed to microcrystalline cellulose (Funacel) and rice straw powder. Interaction between RjCel5B and RjCipA, and RjXyn10C and RjCipA were confirmed by qualitative assays. When a fixed amount of RjCel5B was mixed with different amounts of RjCipA, i.e., at the molar ratio of 6:1 or 6:6, the 6:6 complex showed 6.6-fold higher activity toward Funacel and 11.5-fold higher activity toward rice straw powder than RjCel5B, whereas the 6:1 complex showed only 2.8- and 3.9-folds higher activities toward Funacel and rice straw powder, respectively, than RjCel5B. These results suggest that the family-3 carbohydrate binding module (CBM3) of RjCipA in the RjCel5B-RjCipA complex plays an important role for hydrolysis of cellulose and the substrate-targeting effect of the CBM is more significant than the proximity effect caused by the presence of plural catalytic subunits adjoining each other. In contrast, the 6:1 complex of RjXyn10C and RjCipA showed 45% and 28% of the activities of RjXyn10C toward insoluble wheat arabinoxylan and rice straw powder, respectively. These results suggest that both a negative proximity effect and substrate-isolating effect, but not substrate-targeting effect, are caused by the CBM3 with inappropriate polysaccharide specificity. Substrate-targeting, proximity and substrate-isolating effects are discussed. [ABSTRACT FROM AUTHOR]

Highlights: [•] An essential role of a CBM in substrate recognition by mannanase CtMan5A is reported. [•] A catalytic domain and a CBM collectively form a substrate-binding site. [•] The CBM directly participates in substrate recognition for catalytic action. [•] CtMan5A and its derivative without the CBM yielded different hydrolysis products. [•] Substrate binding modes of CtMan5A and the truncated derivative are different. [Copyright &y& Elsevier]

Abstract: The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-d-glucurono-d-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-l-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan. [Copyright &y& Elsevier]

Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass. [ABSTRACT FROM AUTHOR]

The article presents a study which analyzed the cellulase gene cluster of Clostridium josui which contains man5A gene and the characteristics of recombinant enzyme (rMan5A). The study uses bacterial strains, DNA sequencing, and enzyme assays to determine the cellulase gene cluster of C. josui and the attributes of rMan5A. The results show that the c. josui man5A has encoded a mannanase, and rMan5A has high activity on carob galactomannan and has low activity on guar gum.

Abstract: In previous work, we reported on the identification and purification of a multi-enzyme complex (MEC) from Bacillus licheniformis SVD1. The predominant activity within the MEC was xylanase activity and this study examined the effect of various environmental parameters such as pH, temperature, substrate concentration and compounds such as Mg2+, Mn2+, Fe2+, Zn2+, Ca2+, EDTA, SDS, xylose, xylobiose and ethanol on complexed xylanase activity. The pH optimum was found to be between pH 6.0 and 7.0 and the temperature optimum at 55°C. High levels of residual activity were present over a broad range of pH values. Enhancement of complexed xylanase activity was found in the presence of Mg2+ at 2mM and 10mM, while Ca2+ displayed a slight activation at 2mM but inhibition at 10mM. Mn2+, Fe2+, Zn2+, EDTA and SDS all displayed an inhibitory effect on complexed xylanase activity, with the greatest inhibition found in the presence of Mn2+. Xylose and xylobiose were found to enhance complexed xylanase activity up to 50%, which has not been reported in literature previously. Ethanol was found to inhibit complexed xylanase activity in a competitive manner, but 58% residual activity was still present at concentrations of 50g/l ethanol. Complexed xylanases from B. licheniformis SVD1, being uninhibited by products of degradation and only mildly inhibited by ethanol, would be suitable for use in biotechnological applications such as bioethanol production. [ABSTRACT FROM AUTHOR]

Abstract: Micro-organisms that degrade plant biomass require a range of enzymes to effectively degrade such substrates into sugar monomers. Some organisms secrete a host of free, extracellular enzymes while others produce a multi-enzyme complex (MEC) consisting of a variety of these enzymes. This study examined the cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 with respect to the presence of key enzymes within the MEC found in this organism. When cultured on birchwood xylan, the MEC in B. licheniformis SVD1 was found to contain two endoglucanases (at 25–27kDa, 30–32kDa), seven xylanases (at 20kDa, 38kDa, 42kDa, 45kDa, 48kDa, 54kDa and 70kDa), two mannanases (25kDa, 40–42kDa) and one pectinase (a pectate lyase at 70–72kDa). Identification of enzymes was based on zymogram analysis. The MEC contained 17 different protein species based on SDS-PAGE analysis and 9 of these proteins could be identified through zymograms. Further work will be conducted to confirm the identity of other proteins in the MEC. The crude extract contained a further endoglucanase of 55kDa, a mannanase of 53kDa and a pectin methyl esterase of 38kDa, indicating that not all enzymes are incorporated into the MEC. Understanding the composition of a hemi-cellulolytic system when cultured on a substrate may assist in utilising such a system for the synergistic degradation of complex lignocellulose substrates. [Copyright &y& Elsevier]

Abstract: The cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 was isolated and characterised in birchwood xylan cultures. The predominant activity in the crude culture was xylanase activity, but the crude culture also displayed Avicelase, carboxymethylcellulase (CMCase), mannanase, and pectinase activity. Most of the xylanase activity was found in the culture supernatant, but some activity was cell-associated. Using Sepharose 4B size exclusion chromatography, a 2000kDa multi-enzyme complex (MEC) was purified. The MEC contained predominantly xylanase activity, as well as significant levels of mannanase and CMCase activity, but no Avicelase activity. SDS-PAGE revealed up to eight visible bands in the MEC while zymograms of the MEC displayed two xylanase active bands at 21kDa and 45kDa, and two CMCase active bands at 25kDa and 30kDa. More active bands were visible in the crude supernatant with an additional xylanase active band at 40kDa and an additional CMCase active band at 55kDa. Using thin layer chromatography (TLC), it was established that the crude fraction could release xylose from insoluble birchwood xylan, while the MEC was only able to produce xylobiose from this substrate. The MEC was further able to bind to insoluble xylan, but was unable to bind to crystalline cellulose. This MEC lacks many of the characteristic features of a cellulosome and is most likely a different type of complex. The presence of both high xylanase and mannanase activity makes this MEC unusual. [Copyright &y& Elsevier]

The article explores brachybacterium strain produces a malto-oligosaccharide-forming amylase. The structural gene encoding the amylase from strain was cloned and sequenced. The enzyme hydrolyzed starch to produce maltotriose primarily. This content improves product selectivity in water-miscible organic solvents.

The article discusses the study on anaerobic ammonium-oxidizing bacteria diversity and distribution in Ago Bay sediments in Japan. It states that identified gene sequence in sediment samples establishes a new branch of the anammox group related to anaerobic ammonium oxidizing bacterial sequences. Furthermore, the results showing the presence of anammox-related bacterial clones in five sites of the bay suggest that the bacterium occurs in natural sea sediments.

The article discusses a study which characterized the dihydrolipoyl dehydrogenase in the diaphorase activity of Clostridium kluyveri. It conducted ultraviolet-visible adsorption spectrum and thin layer chromatography analysis for the characterization of bfmBC gene. The enzyme contained a noncovalently but tightly attached flavin adenine dinucleotide (FAD) molecule. The recombinant bfmBC gene catalyzed the oxidation of dihydro-lipoamide with nicotinamide AD as a specific electron acceptor.

The article reports on results of a study of cloning and expression of a Clostridium klyveri gene responsible for diaphorase activity. A description of the study design and method is presented. The study concluded that recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium.

In the global ocean nitrogen cycle, the anaerobic ammonium-oxidizing (anammox) process is recognized as important. In this study, we established an enrichment culture of marine anammox bacteria (MAB) in a column-type reactor. The reactor, which included a porous polyester non-woven fabric that had been placed at the sea floor in advance for enrichment, was continuously fed with NH4Cl and NaNO2 for more than 1 year. Anammox activity in the MAB reactor was confirmed by 15N tracer analysis using 15NH4Cl and Na14NO2. We identified two 16S rRNA genes in the amplified DNA fragments derived from MAB, which were highly homologous with those from Candidatus “Scalindua wagneri” and an uncultured planctomycete clone. Fluorescence in situ hybridization analysis using an anammox-specific probe also confirmed that MAB predominated in the reactor. To our knowledge, this is the first report on the establishment of an enrichment culture of anammox bacteria from the marine environment using a continuous culture system. [ABSTRACT FROM AUTHOR]

The article discusses the study comparing the generation of hydrogen gas in the Refuse-derived fuels (RDF) pellets of Japan and Germany to determine its difference. It was stipulated that RDF pellets of Japan generated large amount of hydrogen gas during its fermentation under wet condition while the Germany RDF pellets did not generate such because of the application of biodegradation process (or biological drying) that reduces the BOD in the garbage. The methods of the study are presented.

The article discusses the result of the study regarding the detection of hydrogen gas-producing anaerobes in refuse-derived fuel (RDF) pellets, in Japan. Investigators analyzed the bacterial cell numbers of RDF samples made with different concentrations of calcium hydroxide. They have determined the amount of hydrogen gas produced under wet conditions. The RDF samples contained different kinds of clostridia capable to produce hydrogen gas, by analyzing its microflora before and during fermentation.

The article presents study that determines the properties of Paenibacillus curdlanolyticus intracellular xylanese Xyn10B encoded by xyn10B gene. It is investigated through the construction of a gene library using Fosmid pCC1FOS and Escherichia coli, wherein the genomic DNA was sheared and separated by pulse-filed electrophoresis. It concludes that the enzyme properties of Xyn10B is different from XynX and XynBE18 although P. curdlanolyticus is homologous to E18 XynBe18 and Aeromonas caviae XynX.

The article reports on results of a study of two proteins with diaphorase activity from Clostridium thermocellum and Moorella thermoacetica. A description of the study design and method is presented. The study concluded two high-molecular-weight rubredoxin genes from Clostridium thermocellum and Moorella thermoacetica were expressed in Escherichia coli and their translated products.

• RjFae1A is a feruloyl esterase containing a CE1 catalytic module and a family-6 CBM. • RjFae1A had higher activity on natural substrate than the CE1 polypeptide. • CBM6 played an important role for release of ferulic acid from natural substrate. • CBM6 interacted with xylose and nonreducing end xylopyranosyl residue of saccharides. • CBM6 preferred wheat arabinoxylan to rye arabinoxylan and beechwood xylan. Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α- l -arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation. [ABSTRACT FROM AUTHOR]

CeIB (BH0603) from Bacillus halodurans is a modular glycoside hydrolase with a family S catalytic module, an immunoglobulin-like module, and module PfamB of unknown function. The recombinant PfamB module bound to Avicel and was essential for CeIB hydrolytic function. We propose that module PfamB be designated a new carbohydrate-binding module. [ABSTRACT FROM AUTHOR]

Studies the hydrogen gas generation from refuse-derived fuel (RDF) under wet conditions. Presence of bacteria that can ferment RDF pellets; Bacteria in the RDF samples fermenting them to generate heat and hydrogen gas.

Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus , with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing. [ABSTRACT FROM AUTHOR]

The article describes the high-level secretion of Bacillus halodurans recombinant xylanase Xyn11A in the yeast Kluyveromyces lactis and its functional properties. According to the authors, the choice of strain background, vector system and promoter for heterologous protein expression in the yeast is of particular importance in terms of yield maximization. They add the Xyn11A showed a specific activity of 628 U/mg-protein on birchwood xylan, but no cellulase or β-xylosidase activity.

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP. [ABSTRACT FROM AUTHOR]

The article presents a study on clostridium stercorarium and its enzymatic components in Japan. The bacteria's modular enzyme is basically composed in 2 hypothetical families of a polysaccharide lyases, the catalytic modules 9-1 (CM9-1) and CM9-2. In the study, the modular enzymes respond to bonding of calcium ions and showed high activity to polygalacturonic acid but revealed lesser reaction towards pectin.

Cites a study determining the role played by family-22 carbohydrate-binding molecule (CBM) in Clostridium thermocellum xylanase Xyn10C. Identification of CBM as a family-10 catalytic molecule of the glycosidase hydrolases responsible for cellulosome assembly from the N-terminus; Ability of recombinant CBM to show an affinity for amorphous celluloses and insoluble and soluble xylan or β-1,3-1,4-glucan; Contribution of Xyn10C in the binding of the enzyme to the substrates and in the stability of the catalytic module polypeptide in the presence of the substrate.

Clostridium stercorarium Xyn10B is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules consecutively from the N-terminus. To investigate the role of the family-22 CBMs, truncated proteins were constructed: a recombinant catalytic module polypeptide (rCD), a CBM polypeptide composed of two family-22 CBMs (rCBM) and a polypeptide composed of the family-22 CBMs and the catalytic module (rCBM-CD). We found that rCBM-CD was highly active toward β-1,3-1,4-glucan; however, rCD was negligibly active toward the same substrate. The Vmax/Km value of rCBM-CD for β-1,3-1,4-glucan was 7.8 times larger than that for oat-spelt xylan, indicating that rCBM-CD should be specified as a β-1,3-1,4-glucanase rather than a xylanase despite the fact that family-10 catalytic modules are well-known xylanase modules. These results indicate that the family-22 CBMs in rCBM-CD are essential for hydrolysis of β-1,3-1,4-glucan. [Copyright &y& Elsevier]

Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 carbohydrate-binding module, two fibronectin type-3 domains, and a family 3 carbohydrate-binding module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli. Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite − 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications. Key points: • PcAA10A is a novel modular LPMO family 10 from Paenibacillus curdlanolyticus. • PcAA10A showed broad substrate specificity on β-1,4 glycosidic linkage substrates. • Non-catalytic domains are important for degrading complex polysaccharides. • PcAA10A is a unique LPMO capable of enhancing lignocellulosic biomass degradation. [ABSTRACT FROM AUTHOR]

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements. [ABSTRACT FROM AUTHOR]

Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors. [ABSTRACT FROM AUTHOR]

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α- l -arabinofuranosidase Axh43A indicated that U1 was α- l -Ara f -(1 → 2)-[α- l -Ara f -(1 → 3)]- d -Xyl p and U2 was α- l -Ara f -(1 → 2)- d -Xyl p , suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the −1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite −1. This is the first report of a xylanase capable of producing α- l -Ara f -(1 → 2)-[α- l -Ara f -(1 → 3)]- d -Xyl p from highly arabinosylated xylan. [ABSTRACT FROM AUTHOR]

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more non-catalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocel-lum hypothetical protein Cthe_0821, defined here as C. thermocellum ManSA, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dock-erin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermo-cellum ManSA hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted &bgr;-l,4-mannoside linkages. The CBM32 of C. thermocellum ManSA displays a preference for the nonreducing ends of mannooligosaccharides, al-though the protein module exhibits measurable affinity for the termini of &bgr;-l,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum ManSA against insoluble mannans but has no significant effect on the capac-ity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum ManSA is affected by the presence of CBM32. [ABSTRACT FROM AUTHOR]

Clostridial cellulosomes are cellulolytic complexes that are formed by highly specific interactions between one of the repeated cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Although Clostridium thermocellum Xyn11A dockerin has a typical C. thermocellum dockerin sequence, in which two amino acid residues are species specifically conserved within the two segments of the dockerin modules, it can recognize Clostridium josui cohesin modules in a non-species-specific manner. The importance of these two conserved amino acids, which are part of the recognition site of the cohesin and dockerin interaction, was investigated by introducing mutations into the first and/or the second segments of the Xyn11A dockerin. Mutations in the first segment did not affect the interactions between dockerin and C. thermocellum and C. josui cohesins. However, mutations in the second segment prevented binding to cohesin proteins. A second round of mutations within the first segment re-established the affinity for both the C. thermocellum and the C. josui cohesins. However, this was not observed for a 'conventional' dockerin, Xyn10C. These results suggest that the combination of the first and second dockerin segments is important for the target recognition. [ABSTRACT FROM AUTHOR]

The article presents a study on the application of the gene encoding xylanase B (xynB) from Clostridium stercorarium, which belongs to family 10 glycosylhydrolases in transgenic plants such as rice and tobacco. It mentions the use of xylanases activity for enzymatic degradation while maintaining cell-free extracts of the plants to continue its normal growth. Results showed that the stable expression of xylanases can improve biofuel and crop production.

Cellulosomes are cellulolytic complexes produced by anaerobic bacteria, and are composed of a scaffolding protein and several catalytic components. The complexes are formed by highly specific interactions of one of the reiterated cohesin modules of the scaffolding protein with a dockerin module of the catalytic components. The affinities of a dockerin module of Clostridium thermocellum CelJ (Cel9D-Cel44A) for several cohesin modules from C. thermocellum and Clostridium josui scaffolding proteins were quantitatively measured by surface plasmon resonance analysis. The recombinant CelJ dockerin-containing protein interacted with three recombinant C. josui cohesin proteins as well as recombinant C. thermocellum cohesin proteins beyond the so-called ‘species specificity’ of the dockerin and cohesin interactions. However, this protein did not recognize a second cohesin module from the C. josui scaffolding protein, suggesting that the catalytic components are not necessarily arranged randomly on a scaffolding protein in native cellulosomes. [ABSTRACT FROM AUTHOR]

The article discusses results of a study investigating the binding specificities of S-layer homology (SLH) module polypeptides derived from the S-layer proteins slp1, Slp2 and cellulosome anchoring protein AncA of the bacterium Clostridium thermocellum. An interaction between peptidoglycan and rSlp1-SLH and rSlp2-SLH was observed. There was no interaction between rSlp1-SLH and rSlp2-SLH and secondary cell wall proteins.

The article investigates the functions of family-22 carbohydrate-binding modules (CBM) in clostridium josui Xyn10A. Enzymes and binding assays characterizes recombinant proteins. Glycoside hydrolases sorts into families based on the amino acid sequence similarities of their catalytic modules. The primary role of CBM is to place the enzyme on the substrate, resulting in an increase in substrate concentration around the enzyme.

Investigates the function of family-9 and family-22 carbohydrate-binding-modules (CBM) in a modular β-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B. Comparison of the enzymatic properties of truncated enzyme of two family-22 CBM and the catalytic module; Role of the addition of family-22 CBM to rCM and rCM-CBM9 in shifting the optimum temperature.

A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs. [ABSTRACT FROM AUTHOR]