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The PHT1 promoter::GUS fusion gene was constructed and introduced into Arabidopsis and rice by Agrobacterium-mediated transformation. Strong beta-glucuronidase (GUS) activity was detected in roots and showed phosphate starvation induction both in Arabidopsis and rice. In contrast, GUS activity in aerial tissues such as those of the leaf and stem was low. In situ GUS staining of root tissue indicated that PHT1 was expressed in root hairs and the outer layer of the main roots, but not in root tips. The PHT1 promoter has a desirable character for biotechnological transgene expression in monocot rice plants.

Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and beta-1,3-1,4-glucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and beta-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially beta-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 degrees C to 75 degrees C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.

Clostridium thermocellum xylanase Xyn10C (formerly XynC) is a modular enzyme, comprising a family-22 carbohydrate-binding module (CBM), a family-10 catalytic module of the glycoside hydrolases, and a dockerin module responsible for cellulosome assembly consecutively from the N-terminus. To study the functions of the CBM, truncated derivatives of Xyn10C were constructed: a recombinant catalytic module polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBM and CM (rCBM-CM). The recombinant proteins were characterized by enzyme and binding assays. Although the catalytic activity of rCBM-CM toward insoluble xylan was four times higher than that of rCM toward the same substrate, removal of the CBM did not severely affect catalytic activity toward soluble xylan or beta-1,3-1,4-glucan. rCBM showed an affinity for amorphous celluloses and insoluble and soluble xylan in qualitative binding assays. The optimum temperature of rCBM-CM was 80 degrees C and that of rCM was 60 degrees C. These results indicate that the family-22 CBM of C. thermocellum Xyn10C not only was responsible for the binding of the enzyme to the substrates, but also contributes to the stability of the CM in the presence of the substrate at high temperatures.

Clostridium josui xylanase Xyn10A is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module (CM), a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. To study the functions of the family-22 CBMs, truncated derivatives of Xyn10A were constructed: a recombinant CM polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBMs and CM (rCBM-CM). Recombinant proteins were characterized by enzyme and binding assays. rCBM-CM showed the highest activity toward xylan and weak activity toward some polysaccharides such as barley beta-glucan and carboxymethyl-cellulose. Although rCBM showed an affinity for insoluble and soluble xylan as well as barley beta-glucan and Avicel in qualitative binding assays, removal of the CBMs negligibly affected the catalytic activity and thermostability of the CM.

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.

The gene encoding the catalytic domain of thermostable xylanase from Clostridium thermocellum F1 was expressed in rice plants under the control of a constitutive promoter. The gene encoding Xylanase A was modified to encode the catalytic domain of family 11 xylanase without the signal sequence (xynA1), and was introduced into rice plants and expressed under the control of a modified cauliflower mosaic virus 35S promoter. Zymogram analysis indicated that the recombinant xylanase was produced in rice plants. The xynA1 gene was stably expressed in rice straw and seed grains. No phenotypic effect of xylanase expression was noted. The enzyme was detected in the desiccated grain. High levels of enzyme activity were maintained in the cell-free extract during incubation at 60 degrees C for 24 h. The results indicated that high levels of xylanase can be produced in rice plants.

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.

A β- N -acetylglucosaminidase gene ( nag 84A) 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 K m and V max 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.

The concentration of estrogenic substances in water and sediment was surveyed in Ago Bay, which is a typical enclosed sea area. Eight water samples and seven sediment samples were collected from the mouth of the Maekawa River at the entrance of the Ago Bay. To examine estrogenic substances, a yeast screen assay developed by Prof. J. P. Sumpter was used. The highest concentrations of estrogenic substances in water and sediment were noted in the Maekawa River (16.6ng·l-1 and 24.3ng·g-1). The concentrations gradually decreased towards the entrance of Ago Bay from the mouth of the Maekawa River. It was suggested that this decrease may be the result of dilution, adsorption to the seabed and biodegradation of the estrogenic substances. The water and sediment assay samples were fractionated by high pressure liquid chromatography and assayed to determine the chemical species of estrogenic substances (high pressure liquid chromatography-bioassay detection method). The estrogenic substances detected corresponded to E1 and/or E2, and were noted in all samples. These are typical natural female hormones, and their origin is presumably domestic sewage and other wastewater containing feces and urine.

Two distinct domains of the xynA gene from Clostridium thermocellum encoding a xylanase catalytic domain (XynAl) and a xylanase catalytic domain with a cellulose binding domain (XynA2) under the control of the cauliflower mosaic virus 35S promoter were electroporated into cultured tobacco BY-2 cells. Transgenic BY -2 calli expressing xylan-hydrolyzing activity were obtained at high frequency for both genes. Western blot analysis using an anti-XynA antibody indicated that XynAl and XynA2 were produced in these calli.

The alpha-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786. Aga36A is an enzyme classified in family 36 of the glycoside hydrolases and showed sequence similarity with some enzymes of family 36 such as Geobacillus (formerly Bacillus) stearothermophilus GalA (57%) and AgaN (52%). The enzyme purified from a recombinant E. coli is optimally active at 70 degrees C and pH 6.0. The enzyme hydrolyzed raffinose and guar gum with specific activities of 3.0 U/mg and 0.46 U/mg for the respective substrates.

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.

The kinetic parameters of Cex, a family 10 xylanase from Cellulomonas fimi , were determined at various pH levels using soluble birchwood xylan (BWX) as a natural polymeric substrate along with three other synthetic aryl-β- d -xylobioside substrates. Using BWX, a high level of substrate inhibition was observed which increased with decreasing pH. In contrast, typical Michaelis-Menten-type profiles were obtained using the three aryl-β- d -xylobiosides as substrates. The k cat values determined using o -nitrophenyl-β- d -xylobioside did not change as the pH increased, whereas the k cat values obtained with BWX, phenyl-β- d -xylobioside and p -nitrophenyl-β- d -xylobioside decreased, suggesting that the presence of an ortho nitro group affects the activity displayed by Cex. These differences were not observed with XynB from Clostridium stercorarium F9, a member of the same family of xylanases as Cex. These results indicate that a careful evaluation is required when employing substituted aryl-β- d -xylobiosides in the characterization of xylanases.

Cellulases catalyze the hydrolysis of cellulose, the major constituent of plant biomass and the most abundant organic polymer on earth. Cellulases are modular enzymes containing catalytic domains connected,vialinker sequences, to noncatalytic carbohydrate-binding modules (CBMs). A putative modular endo-β-1,4-glucanase (BhCel5B) is encoded at locus BH0603 in the genome ofBacillus halodurans. It is composed of an N-terminal glycoside hydrolase family 5 catalytic module (GH5) followed by an immunoglobulin-like module and a C-terminal family 46 CBM (BhCBM46). Here, the crystallization and preliminary X-ray diffraction analysis of the trimodularBhCel5B are reported. The crystals ofBhCel5B belonged to the orthorhombic space groupP21212 and data were processed to a resolution of 1.64 Å. A molecular-replacement solution has been found.

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-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp and U2 was α-L-Araf-(1→2)-D-Xylp, 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-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp from highly arabinosylated xylan.

Plant cell-wall polysaccharides offer an abundant energy source utilized by many microorganisms, thus playing a central role in carbon recycling. Aerobic microorganisms secrete carbohydrate-active enzymes (CAZymes) that catabolize this composite structure, comprising cellulose, hemicellulose and lignin, into simple compounds such as glucose. Carbohydrate-binding modules (CBMs) enhance the efficacy of associated CAZYmes. They are organized into families based on primary-sequence homology. CBM family 46 contains more than 40 different members, but has yet to be fully characterized. Here, a recombinant derivative of the C-terminal family 46 CBM module (BhCBM46) ofBacillus haloduransendo-β-1,4-glucanase B (CelB) was overexpressed inEscherichia coliand purified by immobilized metal-ion affinity chromatography. Preliminary structural characterization was carried out onBhCBM46 crystallized in different conditions. The crystals ofBhCBM46 belonged to the tetragonal space groupI4122. Data were collected for the native form and a selenomethionine derivative to 2.46 and 2.3 Å resolution, respectively. TheBhCBM46 structure was determined by a single-wavelength anomalous dispersion experiment usingAutoSolfrom thePHENIXsuite.

Many investigations were conducted to characterize the cohesin-dockerin interactions. These studies include measuring cohesin-dockerin affinity, identifying critical amino acid residues by site-directed mutagenesis, and determining the molecular structures of the dockerin, cohesin, and its complex. Cohesins are highly conserved within the same scaffolding protein, with sequence identities higher than 50%. The interactions between the cohesin of the Clostridium thermocellum CipA scaffolding protein and the dockerin of cellulosomal catalytic components are categorized as type I, and the interactions between the dockerin of CipA and its counterpart are categorized as type II. The type I cohesins and dockerins include those modules from different microorganisms. This classification, however, is based on sequence homology and does not necessarily imply recognition among the same type of modules. Indeed, interspecies specificity was demonstrated using cohesins and dockerins from C. thermocellum and Clostridium cellulolyticum, respectively. In this work, Pages et al. found that C. thermocellum Cel48A (i.e., CelS), with its dockerin, did not recognize cohesin 1 of the C. cellulolyticum scaffolding protein CipC. The extremely high affinity between cohesin and dockerin and their important roles in cellulosome assembly have prompted interest in determining their molecular structures to elucidate the molecular mechanism of the cohesin-dockerin recognition. The first success in X-ray crystallography of a cohesin-dockerin complex was brought about by using a crystal of the dockerin of C. thermocellum Xyn10B and cohesin 2 of CipA, coexpressed in Escherichia coli and purified as a complex, yielding a good crystal suitable for X-ray analysis.

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.

The cells of Clostridium stercorarium F-9 grown on cellobiose bound to insoluble cellulose allomorphs such as phosphoric acid-swollen cellulose (ASC). Treatment of the cells with 3 M guanidine hydrochloride extracted surface-layer proteins from the cells and abolished the affinity of the cells for ASC. SDS-polyacrylamide gel electrophoresis, zymogram, and immunological analyses indicated that one of the major surface layer proteins was Xyn10B, which is a modular xylanase comprising two family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, and two S-layer homologous (SLH) domains. The C. stercorarium F-9 cells treated with guanidine hydrochloride coprecipitated with ASC upon the addition of a derivative of Xyn10B containing both a CBM and SLH domain in addition to a catalytic domain, but not a derivative without Xyn10B-SLH domains, suggesting that Xyn10B functioned as a cellulose-binding protein in C. stercorarium F-9.

The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.

The chitinolytic bacterium Clostridium paraputrificum strain M-21 produced 2.2 and 1.5 mol hydrogen gas from 1 mol N-acetyl-d-glucosamine (GlcNAc) and ball-milled chitin equivalent to 1 mol of GlcNAc, respectively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment. Waste from the starch industry was also converted to hydrogen. When C. paraputrificum M-21 was cultivated on ball-milled chitin and ball-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or ChiB were detected as the major chitinase species in the supernatant of the cultures, suggesting that the play a critical role in the degradation of chitinous materials.

INTRODUCTIONConjugal plasmids of various incompatibilitygroups avoid restriction when transferred among var-ious bacterial species and genera. This is mainly dueto the ard ( a lleviation of r estriction of D NA) geneswhich are in the leader region of a plasmid and codefor antirestriction proteins [1–5]. The Ard proteins(ArdA, ArdB, ArdC, ArdU, etc.) are small (140–180amino acid residues), highly acidic (total charge –7 to–30), and nonhomologous to each other, sharing onlyan antirestriction motif [6–9]. The motif is commonlyin the C domain, 30–35 residues away from the C end[10]. Its core consists of nine amino acid residues,with a certain arrangement of negatively charged ones(commonly D/E, highly polar N/Q in one site of ArdBand ArdC) and hydrophobic residues located in thecenter and at the ends (Table 1).We have previously shown that restriction allevia-tion induced by conjugal plasmid R64 (incI1) in Escherichia coli K12 is due to arsR , which codes fora suppressor of the ars operon conferring resistance toarsenite and arsenate [13]. ArsR consists of 117 aminoacid residues, is nonhomologous to the Ard proteins,and contains the antirestriction motif 25–35 residuesaway from the N end [13]. Its antirestriction activity islower than in the Ard proteins: ArsR alleviates restric-tion of nonmodified phage λ DNA by a factor of 20,whereas the factor is about 10

The Paenibacillus curdlanolyticus xyn10B gene encoding a family-10 xylanase was cloned and expressed in Escherichia coli, and the recombinant enzyme rXyn10B was characterized. Immunological analysis suggested that Xyn10B is an intracellular enzyme. rXyn10B hydrolyzed birch-wood xylan and xylooligosaccharides to produce mainly xylobiose, suggesting that it is an endoxylanase. Its properties were significantly different from those of some homologous enzymes.

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family xylanases from fungi.

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58 degrees C. It had a Km of 5.1 mg/ml and a Vmax of 123 micromol/min/mg when birch wood xylan was used as a substrate.

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74,511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49,883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-II xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome.

Immediately (17 bp) upstream of the Clostridium paraputrificum chiB gene [J. Bacteriol. 179: 7306-7314 (1997)], we found another chitinase gene chiA encoding chitinase A (ChiA). The chiA gene consists of an open reading frame of 2496 nucleotides and encodes 832 amino acids with a deduced molecular mass of 92,585 Da. The mature ChiA is a modular enzyme composed of a family-18 catalytic domain responsible for chitinase activity, two cadherin-like domains, and a chitin-binding domain. The domain organization of ChiA is fundamentally identical to that of ChiB and the overall sequence identity between them is 35.4%. ChiA was purified from the periplasm fraction of Escherichia coli harboring the chiA gene. The molecular mass of purified ChiA (89,000 Da), determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, was in good agreement with the value (89,119 Da) calculated from the deduced amino acid sequence, excluding the signal peptide. Immunological and N-terminal amino acid sequence analyses revealed that ChiA and ChiB are major chitinases of C. paraputrificum and their production is inducible by ball-milled chitin. Northern blot analysis indicated that the chiA and chiB genes constitute a polycistronic operon. Primer-extension analysis confirmed that the transcription of this operon starts upstream of chiA.

Saccharomyces cerevisiae rhk mutants were previously shown to have a phenotype that is resistant to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895. The RHK1/ALG3 gene encodes a mannosyl-transferase that is involved in the synthesis of an oligosaccharide in protein N-glycosylation. Previously, this gene was cloned and shown to complement the rhk1 mutation. In this study, the RHK2 gene, which complements the rhk2 mutation, was cloned. The RHK2 gene was found to be identical to the essential gene STT3, which encodes a subunit of the oligosaccharyl-transferase complex. This complex transfers the core oligosaccharide to proteins. The rhk2 mutants showed supersensitivity to several drugs (Calcofluor White, caffeine and FK506), suggesting that these strains have cell-wall defects. Activity staining of invertase in an acrylamide gel indicated that it was underglycosylated. These results suggest that one or more mannoproteins are involved in the cytocidal process of HM-1.

Transgenic tobacco plants exhibiting exogenous cellulase activity were produced by introduction of a truncated cellulase (t-EgI) gene from Ruminococcus albus . They accumulated about 0.1–0.5% t-EgI of the total soluble proteins in the cytoplasm. They were normal in morphology and growth. t-EgI expressed in the transgenic plants degraded their own cell wall materials after the cells were disrupted. The plants expressing the t-egI gene could be used as “cellulase-encapsulating plants” for livestock and food industries.

Two high-molecular-weight rubredoxin genes (hrb) from Clostridium thermocellum and Moorella thermoacetica were expressed in Escherichia coli and their translated products (Hrb) were characterized. M. thermoacetica Hrb showed strong diaphorase activity. In contrast, C. thermocellum Hrb, containing neither FMN nor FAD in the molecule, showed flavin reductase activity with moderate diaphorase activity. Both enzymes were optimally active at about 50 degrees C.

We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.

The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of C. josui CipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticum CipC, but C. josui CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of the C. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.

The chiA gene encoding chitinase A was cloned into Escherichia coli from Xanthomonas sp. strain AK and its nucleotide sequence was determined. The structural gene consists of 1788 bp encoding 596 amino acids with a predicted molecular weight of 62,122. The deduced ChiA is a modular enzyme composed of an N-terminal signal peptide and four domains in the following order: a chitin-binding domain, two fibronectin type III domains, and a family 18 catalytic domain. ChiA purified from the recombinant E. coli had temperature and pH optima at 35°C and 4.5, respectively. The Km and Vmax values for colloidal chitin were estimated to be 1.8 mg/ml and 8.7 μmol/min/mg, respectively.

Xylanase A (XynA) from Clostridium stercorarium , which consists of a catalytic domain and two family VI cellulose-binding domains (CBDs) each connected by a linker sequence, was found to bind to insoluble oat-spelt xylan as well as acid-swollen cellulose (ASC). Its adsorption to xylan was greatly dependent on the concentration of the phosphate buffer in the binding assay mixtures. The adsorption of XynA to insoluble xylan proceeded in accordance with a Langmuir-type isotherm. The Ka and [PX] max values of XynA for oat-spelt were 0.17 l /μmol and 5.0 μmol/g, respectively. Removal of the CBDs from XynA abolished the cellulose- and xylan-binding abilities of this enzyme and reduced the enzyme activity toward insoluble xylan but not soluble xylan. These results clearly indicated that the CBDs of XynA play an important role in the hydrolysis of insoluble xylan. Desorption of XynA from the ASC-XynA complex was effected by soluble saccharide solutions such as barley β-glucan, birchwood xylan, and glucose solutions as well as a cellobiose solution, indicating that the CBDs of XynA have an affinity for soluble saccharides in addition to insoluble polysaccharides.

The complete nucleotide sequence of a new cryptic plasmid pAR67 isolated from a rumen bacterium Ruminococcus albus AR67 has been determined. The plasmid pAR67 was 3419 bp in size with a 45% GC content. Two open reading frames, ORF1 and ORF2, encoding potential polypeptides of 285 and 165 amino acids, with limited sequence similarity to replication and mobilization proteins, respectively, were identified within the sequence. The region upstream of ORF1 had an AT-rich (80%) segment followed by four 19-bp direct repeats, which is similar to the structural organization characteristic of replication origins of some bacterial plasmids.

The plasmid-encoded arsenical resistance operon of plasmid pKW301 confers a high level resistance to arsenicals and antimonials on cells of Escherichia coli. Although it has been reported that ArsR of pKW301, a homologue of negative regulatory proteins, was previously shown to exhibit only weak homology to other ArsR proteins, it was identified as a DNA-binding protein by its behavior in gel mobility shift assays. In this study, ArsR exhibited a specific affinity for the ars promoter region. Gel mobility shift assay in the presence of sodium arsenite and potassium antimonial tartrate indicated that these compounds were inducers of expression of the ars operon. The specific binding site, as determined by DNase I footprint analysis, spans nucleotides 28 and 31 of the coding and noncoding strand, respectively, in the ars promoter region. ArsD, a second putative repressor protein encoded by arsD, was also identified as a DNA-binding protein by gel mobility shift assay.

A xylanase gene xynG1 was isolated and sequenced from Aspergillus oryzae KBN616 used for making shoyu koji. The structural part of the xynG1 gene was found to be 725 bp and was predicted to be interrupted by a single intron which was 62 bp in size. The mature XynG1 was predicted to be 189 amino acids in size and had high amino acid homology to other fungal xylanases classified in family 11 glycosidase. The XynG1 was detected in the culture supernatant of A. oryzae KBN616 grown in xylan medium but was not detected when A. oryzae KBN616 was grown in glucose medium. The xynG1 gene was introduced into A. nidulans and was found to be expressed even in the glucose media. This expression pattern was confirmed using a luciferase gene as a reporter in A. nidulans.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.

(1997). Structure of Cellulases and Their Applications. Biotechnology and Genetic Engineering Reviews: Vol. 14, No. 1, pp. 365-414.

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall beta-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of beta-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.

The manA gene encoding an extracellular β-1,4-mannanase of a marine bacterium, Vibrio sp. strain MA-138, was cloned and sequenced. The manA gene consists of an open reading frame of 1185 nucleotides encoding a protein of 395 amino acids with a molecular weight of 43,097. A putative ribosome-binding site and a promoter region were identified in the DNA sequence. ManA of Vibrio sp. strain MA-138 was classified into family 5 of the glycosyl hydrolases and is highly homologous to the ManAs of Caldocellum saccharolyticum (sequence identity: 53%) and Streptomyces lividons (sequence identity: 51%). The N-terminal amino acid sequence of the recombinant enzyme was completely in agreement with that deduced from the nucleotide sequence and that of the enzyme purified from strain MA-138. Although themolecular weight of the recombinant ManA was smaller than that of the enzyme from strain MA-138 on SDS-polyacrylamide gel electrophoresis, this was attributed to linkage of the carbohydrate chain to the latter protein. The enzymatic properties of the recombinant ManA was similar to those of the enzyme from strain MA-138.

The gene encoding the single cellulose-binding domain II (CBD II) of Clostridium stercorarium xylanase A was fused to the egIV gene encoding endoglucanase IV (EGIV) from Ruminococcus albus. The fusion protein (EGIV + CBDII) expressed in Escherichia coli can be readily purified from the cell-free extract of E. coli in a single step using the affinity of CBD to cellulose. The purified enzyme was cleaved into two moieties, i.e. the catalytic domain and CBD, at a specific site in the linker region by partial digestion with trypsin at 4°C. This result indicates that this CBD belonging to family VI of CBD families can be used as an affinity tag for purification of the recombinant protein.

The xylanase gene (xynB) from Clostridium stercorarium was expressed in tobacco cells (BY2) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The gene product, xylanase B (XynB), was proteolytically processed in the transformed cells (termed B1-11): a larger form of the protein was mainly found in the culture supernatant and a smaller one was found in the cells. The amount of XynB protein was estimated to be 5% of the total soluble proteins in B1-11 calli after 2 weeks of cultivation. XynB isolated from the transformed tobacco cells had the activity to hydrolyze the cell wall of barley straw.

© 2013 Ratanakhanokchai et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Paenibacillus curdlanolyticus Strain B-6 Multienzyme Complex: A Novel System for Biomass Utilization

The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s−1 mM−1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055. The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative. CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily E1 endoglucanase domain, a family J endoglucanase domain, a docking domain, and another domain of unknown function (UD-2). UD-1 has no significant similarity to UD-2. CelJ hydrolyzed carboxymethylcellulose and xylan, and xylanase activity was ascribed to the family J domain. Antiserum raised against the truncated CelJ cross-reacted with proteins contained in the cellulosome of C. thermocellum F1. These results strongly suggest that CelJ is equivalent to S2, which was identified as the largest catalytic component in the cellulosome of C. thermocellum YS. A second but incomplete ORF encoding an enzyme classified in subfamily E2 endoglucanase, was located downstream of celJ.

The gene (egI) encoding a cellulase (EgI) from Ruminococcus albus was modified for production of the three forms of a cellulase: the pre-form having a signal sequence region, the mature form lacking the signal sequence region and a truncated form, which contained neither the signal sequence nor the N-terminal 15 amino acid residues. These modified genes were expressed in tobacco suspension cells (BY-2) under the control of cauliflower mosaic virus 35S promoter. Among the transformants harboring each type of the gene, cells having the gene encoding the truncated egI showed the strongest cellulase activity. In this transformant, 95% of the cellulase activity was located in cytoplasm and the total cellulase activity was approximately 30 times stronger than the endogenous cellulase activity in the non-transformed tobacco cells. The growth of the transformants harboring the gene encoding the trucated EgI was negligibly affected by the high level of expression of the truncated EgI.