Search

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.

The article reports on results of a study of the effect of carbon sources on the induction of xylanolytic-cellulolytic multienzyme complexes in Paenibacillus curdlanolyticus strain B-6. A description of the experimental set-up and measurement methods is presented. The study concluded that multienzyme complexes of P. curdlanolyticus B-6 consisted of the main enzymes and non-enzymatic subunits.

A novel chitinase gene chiC of Clostridium paraputrificum M-21, a chitinolytic and hydrogen-gas-producing bacterium, was characterized along with its translated product. The chi18C gene encodes 683 amino acids (signal peptide included) with a deduced molecular weight of 74,651. Chi18C is a modular enzyme composed of a family-18 catalytic module of glycoside hydrolases, two reiterated modules of unknown function, and a family-12 carbohydrate-binding module. Recombinant Chi18C was active toward soluble and insoluble chitin preparations, and synthetic substrates such as 4-methylumbelliferyl-β- d- N-N′ -N″-triacetylchitotriose, but not active toward 4-MU- N-acetylglucosamine or 4-MU-β- d- N-N′-diacetylchitobioside. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunological analyses suggested that the expression of chi18C was inducible with chitinous substrates and that Chi18C was secreted into the culture medium. A possible role of Chi18C in the chitinolytic system of C. paraputrificum M-21 is discussed. [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.

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.

The article discusses the result of the study regarding a novel thermophilic pectate lyase containing catalytic modules of Clostridium stercorarium, in Japan. The pectase lyase Pel9A was encoded by C. stercorarium F-9 pel9A gene. The family-9 catalytic modules of polysaccharide lyases include CM9-1 and CM9-2, derived from a modular enzyme, which is the mature form of Pel9A. This enzyme required calcium ion for its activity and showed a higher activity toward polygalacturonic acid but lower toward pectin.

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.

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.

Recent papers concerning the application of microbial genes to recalcitrant biomass utilization and environmental conservation are reviewed. Microbial genes have been integrated and expressed in plants and microorganisms. When cellulose-degrading enzyme genes are expressed in rice plants, the transgenic plants exhibit swollen cell walls which increases the digestibility of rice straw in the rumen. When genes encoding aromatic compound-degrading enzymes are expressed in plants, it is expected that aromatic compounds contaminating soil would be degraded during the growth of the transgenic plants. The former transgenic plants are utilized as feed and the latter for phytoremediation. Dockerin and cohesin interactions occurring in the cellulase complex, cellulosome, are applied to the construction of artificial enzyme complexes and protein purification by expressing the genes in transformed bacteria and/or silkworms, respectively. In the case of the forced expression of bacterial genes encoding chitinase and/or hydrogenase in the wild-type bacteria, chitin degradation and hydrogen gas production in the transformed bacteria occur at much higher rates than in the wild type. [Copyright &y& Elsevier]

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 presents a study on the effect of family 22 carbohydrate-binding module (CBM22) on the thermotolerance of Xylanase10B from Clostridium stercorarium. The derivative scanning of calorimetry showed that CBM22 conferred resistance to thermal unfolding of the enzyme and avoided the enzyme from refolding after thermal unfolding. The thermal activity and the reversibility of thermal unfolding should be checked to clarify the reason for CBM22 thermostabilizing effect.

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]

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96 Å. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a β-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu186 and Glu359, are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a NMR experiment using the reduced cellooligosaccharide as a substrate. [ABSTRACT FROM AUTHOR]

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]

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the KD values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains. [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]