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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]

Carbohydrate binding modules (CBMs) are commonly known to have the potential to increase binding of carbohydrate-active enzymes to target polysaccharides, thus the catalytic module can easily contact the substrate via CBM. Laccase is one of the most important enzymes used for delignification step in paper industry. This enzyme improves the product's quality, industrial process, and environmentally sound technologies. In this study, we have constructed the fusion gene encoding a novel CBM36 from β-Xylosidase B (GbtXyl43B) of Geobacillus thermoleovorans IT-08 and laccase (LCC2) of Pleurotus salmoneostramineus using overlapping polymerase chain reaction (PCR), and inserted into expression vectors pET-32a and pYHM1 for Escherichia coli BL21 codon plus and Saccharomyces cerevisiae BJ1824, respectively. The fusion protein, Cbm36-Lcc2, was successfully expressed both in E. coli and S. ceerevisiae and optimum pH and temperature were determined to be 5.0 and 60 °C, and 6.0 and 60 °C, respectively, with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate; ABTS) as the substrate. The thermostability and pH stability indicated that Cbm36-Lcc2 expressed in S. cerevisiae BJ1824 was more stable than in E.coli BL21 codon plus. The laccase activity for Cbm36-Lcc2 was two to three times higher than that for Lcc2 itself. This study developed the new insight that fusion of novel Cbm36 into Lcc2 could improve the thermostability of Lcc2 in its chimeric laccase form of Cbm36-Lcc2 compared to Lcc2 itself. • Laccase is one of enzymatically innovations for delignification processes that are more efficient and eco-friendly. • The novel CBM36 gene was successfully fused to LCC2 using an overlapping PCR, and could be expressed in heterologous host. • New insight displayed that the chimeric laccase could improve its thermostability and activity than that for Lcc2 itself. [ABSTRACT FROM AUTHOR]

The article presents a study that identifies a catalytic residue of Clostridium paraputrificum N-acetyl-β-D-glucosaminidase 3A (Nag3A), an enzyme classified in family 3 of the glycoside hydrolases. Mutations were introduced into highly conserved residues to identify catalytic residues of such enzyme. Escherichia coli strains, which were grown at 37 degrees celsius and supplemented with appropriate antibiotics, were used as the host for expression of the recombinant proteins from plasmids.

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]

Describes the cloning and sequencing of new Ruminococcus albus endoglucanase genes cel5D and cel9A encoding family-5 and family-9 endoglucanases. Purification of truncated derivatives of cel5D from Escherichia coli recombinants; Presence of a cellulosome; Interaction between the dockerin modules of the catalytic components and the repeated cohesin modules of the scaffolding protein.

Discusses the cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium α-galactosidase Aga36A in Escherichia coli. Nucleotide sequence of aga36A and its flanking regions; Amino acid sequences of Aga36A and other open reading frames; Comparison of the α-Gal gene clusters.

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.

Clostridium paraputrificum M-21 was transformed with several shuttle plasmids constructed for Clostridium acetobutylicum-Escherichia coli and Clostridium perfringens-E. coli by electroporation. The Clostridium stercorarium xylanase gene xyn10B was successfully expressed in C. paraputrificum M-21 and the expressed protein did not suffer from proteolysis by host protease(s). This system will provide us with a genetic tool for genetic and metabolic engineering of this bacterium. [Copyright &y& Elsevier]

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]

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]

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]

A gene encoding an exo-β-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-β-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50°C and catalyzes hydrolysis only of β-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from β-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a β3-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-β-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including 13-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a β3-1,3-galactobiose, β3-1,4-galactosyl glucose, or β3-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward β3-1,3-galactan and α-L-arabinofuranosidase-treated AGP. [ABSTRACT FROM AUTHOR]

A β-N-acetylglucosaminidase gene (nag84A) was cloned from Clostridium paraputrificum M-21 in Escherichia coli. The nag84A gene consists of an open reading frame of 4647 by encoding I549 amino acids, with a deduced molecular weight of 174,311, which have a catalytic domain belonging to family 84 of the glycoside hydrolases. Nag84A was purified from a recombinant E. coli and characterized. Although Nag84A exhibited high homology to the hyaluronidase from Clostridium perfringens, it did not degrade hyluronic acid. The enzyme hydrolyzed chitooligomers such as di-, tri-, tetra-, penta- and hexa-N-cetylchitobexaose, and synthetic substrates such as 4-methylumbelliferyl N-acetyl β-d-glucosaminide [4-MU-(G1cNAc)], but did not hydrolyze 4-MU-β-d-glucoside, 4-MU-α-d-glycoside, 4-MU-α-d-GlcNAc, 4-MU-α-d-galactoside, 4-MU-β-d-xyloside, PNP-β-d-galactoside, and PNP-α-d-xyloside. The enzyme was optimally active at 50°C and pH 6.5, and the apparent Km and Vmax values for 4-MU-(G1cNAc) were 8.5 μM and 1.39 μmol/min/mg of protein, respectively. SDS-PAGE, zymogram, and immunological analyses suggested that Nag84A was inducible by ball-milled chitin. Since Nag84A has a high molecular weight with a family 84 catalytic domain with high homology to hyaluronidases but no hyaluronidase activity, the enzyme is a novel β-N-acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family I8. [Copyright &y& Elsevier]

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]