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A cellulase gene cluster of Clostridium josuiwas sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josuicellulosome. Among these genes, the man5Agene encoding β-mannanase was expressed in Escherichia coliand the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.

The cbnAgene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutrophaNH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula× P. tremuloides). Integration of the cbnAgene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnAgene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.

A family-4 α-galactosidase Mel4A of Bacillus haloduranswas expressed in Escherichia coliand characterized. Recombinant enzyme rMel4A depended on NAD+, some divalent cations such as Mn2+, and reducing reagents such as dithiothreitol. rMel4A was active on small saccharides such as raffinose but not on highly polymerized galactomannan. Immunological analysis indicated that raffinose induced the production of Mel4A in B. halodurans.

A small enzyme showing diaphorase activity was purified from culture supernatant of Clostridium kluyveriand its N-terminal amino acid sequence was determined. This sequence identified a gene (diaA) encoding a protein (DiaA) of 229 amino acids with a predicted molecular weight of 24,981 in the genomic DNA sequence database of C. kluyvericonstructed by the Research Institute of Innovative Technology for the Earth. The predicted protein was composed of a flavin reductase-like domain and a rubredoxin-like domain from its N-terminus. The diaAgene was cloned into an expression vector and expressed in an Escherichia colirecombinant. Recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium. The enzyme was most active at pH 8.0 at 40 °C. The UV-visible absorption spectrum and thin layer chromatography (TLC) analyses indicated that one rDiaA molecule contained a tightly bound FMN molecule as a prosthetic group. An iron molecule was also detected in an enzyme molecule.

The effect of polymeric substances such as α-cellulose, birchwood xylan, corn hull, and sugarcane bagasse, and of soluble sugars such as L-arabinose, D-galactose, D-glucose, D-xylose, and cellobiose, on the induction of multienzyme complexes in a facultatively anaerobic bacterium, Paenibacillus curdlanolyticusB-6, was investigated under aerobic conditions. Cells and culture supernatants of strain B-6 grown on different carbon sources were analyzed. Cells grown on each carbon source adhered to cellulose. Hence strain B-6 cells from all carbon sources must have an essential component responsible for anchoring the cells to the substrate surfaces. Native–polyacrylamide gel electrophoresis (native–PAGE), sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), zymogram analysis, and enzymatic assays indicated that many proteins having xylanolytic and cellulolytic activities from P. curdlanolyticusB-6 grown on each carbon source were produced as two multienzyme complexes in the culture supernatants. These results indicate that P. curdlanolyticusB-6 produced multienzyme complexes when grown on both polymeric and soluble sugars. The multienzyme complexes of P. curdlanolyticusB-6 consisted of the main enzymes and non-enzymatic subunits and the production of some different subunits, depending on the carbon source.

The putative xyn11Astructural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus haloduransstrain C-125 was heterologously expressed in the yeast Kluyveromyces lactisCBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactisCBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.

S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellumS-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellumcell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia colicells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.

Clostridium paraputrificumM-21 β-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the Kmvalues of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificumNag3A.

Clostridium stercorariumF-9 pectate lyase Pel9A is a modular enzyme composed of two hypothetical family-9 catalytic modules of the polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. In this study, we constructed and characterized CM9-1 and CM9-2 polypeptides as rCM9-1 and rCM9-2 respectively. Both of them, like the full-length Pel9A, required the Ca2+ion for their enzyme activities and showed high activity toward polygalacturonic acid but lower activity toward pectin. The specific activity of rCM9-2 was three times higher than that of rCM9-1 and rCM9-2 by itself efficiently catalyzed the depolymerization reaction of polygalacturonic acid into monosaccharide as the major product. It was found that rCM9-1 and rCM9-2 adsorbed to polygalacturonic acid and pectin on native affinity PAGE analysis, suggesting that they contain an independent carbohydrate-binding module separable from a catalytic module or consist of a catalytic module with a binding affinity for pectic substrates.

Clostridium stercorariumXyn10B having hydrolytic activities on xylan and β-1,3-1,4-gllucan 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 β-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially β-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 °C to 75 °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.

The Clostridium stercorariumF-9 xyl43Bgene encoding the β-xylosidase Xyl43B consists of an open reading frame of 1,491 nucleotides that encodes a putative protein, classified in family 43, of 497 amino acids with a predicted molecular weight of 56,355. The deduced amino acid sequence of Xyl43B has sequence similarity with β-xylosidases from Bacteriodes thetaiotaomicron(57% sequence identity), Prevotella ruminicola(45%), Streptomyces coelicolor(40%), and Clostridium acetobutylicum(36%), all of which have been classified in family 43 of the glycoside hydrolases. Xyl43B was purified from a recombinant Escherichia coliand characterized. The optimum pH of the purified enzyme was 3.5 and it was stable over pH from 3.0 to 8.0. Its optimum temperature was 80°C and it showed thermostability in the temperature range from 50 to 70°C. Xyl43B had a Kmof 6.2 mMand a Vmaxof 15 μmol min−1mg−1for p-nitrophenyl-β-D-xylopyranoside.

An EcoRI chromosomal DNA fragment of Ruminococcus albusF-40 that conferred endoglucanase activity on Escherichia coliwas cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5Dand cel9Arespectively. The cel5Dgene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The cel9Agene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E. coli. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 μmol/min/mg for carboxymethycellulose.

The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellumXynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP=2−8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, Ka, increased with increasing DP from 5×103M−1(DP=2) to approximately 5×105M−1(DP=5-8) at 20°C. The Kavalues at 60°C were about 1/10 of those at 20°C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.

The α-galactosidase gene aga36Aof Clostridium stercorariumF-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36Agene 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) stearothermophilusGalA (57%) and AgaN (52%). The enzyme purified from a recombinant E. coliis optimally active at 70°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.

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzaeKBN616, used for making shoyu koji.The structural part of xynF3was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3gene was successfully overexpressed in A. oryzaeand the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58°C. It had a Km of 6.5 mg/ml and a Vmaxof 435 μmol•min−1•mg−1when birch wood xylan was used as a substrate. Expression of the xynF3gene was analyzed using an Eschelichia coliβ-glucuronidase gene as a reporter. The result indicated that xynF3is expressed in the medium containing wheat bran as a carbon source.

The cells of Clostridium stercorariumF‐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. stercorariumF‐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. stercorariumF‐9.

The Ruminococcus albusF-40 egVgene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albusF-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recounbinant Escherichia coliand characterized since EGV suffered from partial proteolysis by E. coliprotease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40°C, respectively. By Western blot analysis using the antiserum raised against the truncated enzylne, EGV was detected in the whole cells but not in the culture supernatant of R. alubusF-40, suggesting that EGV was located on the cell surface.

The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albusstrain F-40 grown on cellulose by a Sephacryl S-300HR column chromatography. The molecular mass of the cellulolytic complex was found to be larger than 1.5×106Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40 kDa to 250 kDa. Among them, 11 proteins showed endoglucanase and/or xylanase activities on the zymograms. Immunological analysis using an antiserum raised against the dockerin domain of endoglucanase VII of R. albus(DocVII) suggested that at least 7 proteins in the cellulolytic complex contained a dockerin domain immunoreactive with the anti-DocVII antiserum. Furthermore, DocVII was shown to specifically interact with a 40-kDa protein of the cellulolytic complex by Far-Western blot analysis. These results strongly suggest that the cellulolytic complex produced by R. albusresembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellumand respective components are assembled into the cellulosome by the mechanism common in all of the cellulolytic clostridia, i.e., the cellulosome is formed by the interaction between a dockerin domain of catalytic components and a cohesin domain of a scaffolding protein.

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzaeKBN616, used for making shoyu koji. The structural part of xynG2was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2was 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 xynG2gene was successfully overexpressed in A. oryzaeand 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°C. It had a Km of 5.1 mg/ml and a Vmax of 123 μmol/min/mg when birch wood xylan was used as a substrate.

The nucleotide sequence of the Clostridium josuiFERM P-9684 xyn10Agene, 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. josuiFERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia colistrain 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. colistrain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, or carboxymethylcellulose.

The nucleotide sequence of the Clostridium stercorariumF-9 xynCgene, encoding a xylanase XynC, consists of 3,093 bp and encodes a 1,031-amino acids with a molecular weight of 115,322. XynC 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 IX cellulose-binding domain, and two S-layer homologous domains. Immunological analysis indicated the presence of XynC in the culture supernatant of C. stercorariumF-9 and in the cells, most likely on the cell surface. XynC purified from a recombinant E. coliwas highly active toward xylan and slightly active toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, p-nitrophenyl-β-D-glucopyranoside, and carboxymethylcellulose. XynC hydrolyzed xylan and xylooligosaccharides larger than xylotriose to produce xylose and xylobiose. This enzyme was optimally active at 85°C and was stable up to 75°C at pH 5.0 and over the pH range of 4 to 7 at 25°C.

The Clostridium thermocellumendoglucanase CelJ contains two different catalytic domains in a polypeptide, i.e., a subfamily E1 catalytic domain and a family J catalytic domain [J. Bacteriol., 178, 5732–5740 (1996)]. The family J catalytic domain (CDJ-CelJ) was produced by a recombinant Escherichia coliand purified. The purified CDJ-CelJ gave a single band on SDS–polyacrylamide gel electrophoresis and the molecular weight of this enzyme (60,000) was consistent with the value (60,333) calculated from the DNA sequence. CDJ-CelJ hydrolyzed various cellulosic substrates, xylan, and lichenan but not p-nitrophenyl (PNP)-cellobioside, PNP-glucoside, or PNP-xyloside at all. CDJ-CelJ was active on Avicel, a microcrystalline cellulose, and the specific activity of CDJ-CelJ on Avicel (0.0078 U/mg protein) was comparable to that of CelS, which is recognized as the most important catalytic subunit of the C. thermocellum, cellulosome, suggesting that CelJ is also an important catalytic subunit in the cellulosome of this bacterium, in addition to CelS.

The gene (egI) encoding a cellulase (EgI) from Ruminococcus albuswas 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 egIshowed 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.

Restriction endonuclease RalF40I was purified from cell‐free extracts of the rumen cellulolytic bacterium Ruminococcus albusF‐40 by heparin‐Sepharose chromatography. The preparation was active only on DNA substrates that were not Dam‐methylated. RalF40I recognizes the 4‐bp palindrome, 5′‐/GATC‐3′, and cleaves DNA at the 5′ side of G in the sequence, producing 5′ tetranucleotide protruding ends. RalF40I is a class II restriction endonuclease and an isoschizomer of MboI and DpnII.

The Paenibacillus curdlanolyticusxyn10Bgene 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.

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

A cellulolytic and thermophilic anaerobe was isolated from soil. This bacterium made a halo on a roll-tube culture containing Avicel. Analysis of the PCR-based 16S rRNA gene sequence showed that the bacterium was closely related to Moorella thermoacetica. Scanning electron microscopy showed the bacterium is a rod and has no protuberant structure on the surface of cells growing on cellulose, suggesting that this strain is a non-cellulosomal cellulolytic bacterium. Carboxymethyl cellulase and xylanase activities were detected in the culture broth. A major fermentation product from ball-milled cellulose was acetate. This strain has a potential to convert cellulosic biomass to acetate, directly.