Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol

Abstract Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

Resumo Hoje, o foco global da pesquisa é explorar a solução da crise energética e da poluição ambiental. Como em outros países agrícolas, grandes quantidades de cascas de melancia (WMP) são descartadas como resíduos no meio ambiente no Paquistão, mas a gestão adequada desses resíduos é a mais recente solução para salvar o meio ambiente da poluição. O trabalho enfatiza o papel das leveduras etanologênicas para utilizar açúcares significativos presentes no WMP para fermentação de bioetanol de baixo custo. A hidrólise de ácido clorídrico diluído de WMP foi realizada em condições otimizadas empregando RSM (metodologia de superfície de resposta) e seguindo o projeto de composto central (CCD). Este projeto experimental é baseado na otimização da etanologenesis envolvendo alguns parâmetros independentes importantes, como hidrolisado de WMP e razão de meio sintético (X1), temperatura de incubação (X2) e temperatura de incubação (X3) para rendimento máximo de etanol explorando o padrão (Saccharomyces cerevisiae K7) também como leveduras experimentais (Metchnikowia cibodasensis Y34). Os resultados revelaram que os rendimentos máximos de etanol obtidos a partir de S. cerevisiae K7 foi de 0,36 ± 0,02 g / g de açúcares redutores, enquanto M. cibodasensis Y34 rendeu 0,40 ± 0,01 g de etanol / g de açúcares redutores. O isolado de levedura M. cibodasensis Y34 apareceu como um etanologeno promissor e incorpora um potencial prospectivo para a valorização fermentativa de WMP em bioetanol.

Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol / Biotransformação otimizada de hidrolisado de casca de melancia tratada com ácido em etanol

Abstract Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

Resumo Hoje, o foco global da pesquisa é explorar a solução da crise energética e da poluição ambiental. Como em outros países agrícolas, grandes quantidades de cascas de melancia (WMP) são descartadas como resíduos no meio ambiente no Paquistão, mas a gestão adequada desses resíduos é a mais recente solução para salvar o meio ambiente da poluição. O trabalho enfatiza o papel das leveduras etanologênicas para utilizar açúcares significativos presentes no WMP para fermentação de bioetanol de baixo custo. A hidrólise de ácido clorídrico diluído de WMP foi realizada em condições otimizadas empregando RSM (metodologia de superfície de resposta) e seguindo o projeto de composto central (CCD). Este projeto experimental é baseado na otimização da etanologenesis envolvendo alguns parâmetros independentes importantes, como hidrolisado de WMP e razão de meio sintético (X1), temperatura de incubação (X2) e temperatura de incubação (X3) para rendimento máximo de etanol explorando o padrão (Saccharomyces cerevisiae K7) também como leveduras experimentais (Metchnikowia cibodasensis Y34). Os resultados revelaram que os rendimentos máximos de etanol obtidos a partir de S. cerevisiae K7 foi de 0,36 ± 0,02 g / g de açúcares redutores, enquanto M. cibodasensis Y34 rendeu 0,40 ± 0,01 g de etanol / g de açúcares redutores. O isolado de levedura M. cibodasensis Y34 apareceu como um etanologeno promissor e incorpora um potencial prospectivo para a valorização fermentativa de WMP em bioetanol.

Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol.

Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

EEG Artifact Removal in TMS Studies of Cortical Speech Areas.

The combination of transcranial magnetic stimulation (TMS) and electroencephalography (EEG) is commonly applied for studying the effective connectivity of neuronal circuits. The stimulation excites neurons, and the resulting TMS-evoked potentials (TEPs) are recorded with EEG. A serious obstacle in this method is the generation of large muscle artifacts from scalp muscles, especially when frontolateral and temporoparietal, such as speech, areas are stimulated. Here, TMS-EEG data were processed with the signal-space projection and source-informed reconstruction (SSP-SIR) artifact-removal methods to suppress these artifacts. SSP-SIR suppressed muscle artifacts according to the difference in frequency contents of neuronal signals and muscle activity. The effectiveness of SSP-SIR in rejecting muscle artifacts and the degree of excessive attenuation of brain EEG signals were investigated by comparing the processed versions of the recorded TMS-EEG data with simulated data. The calculated individual lead-field matrix describing how the brain signals spread on the cortex were used as simulated data. We conclude that SSP-SIR was effective in suppressing artifacts also when frontolateral and temporoparietal cortical sites were stimulated, but it may have suppressed also the brain signals near the stimulation site. Effective connectivity originating from the speech-related areas may be studied even when speech areas are stimulated at least on the contralateral hemisphere where the signals were not suppressed that much.

Transcranial magnetic stimulation-evoked potentials after the stimulation of the right-hemispheric homologue of Broca's area.

The combination of transcranial magnetic stimulation and electroencephalography can be applied to probe effective connectivity. Neurons are excited by magnetic pulses, which produce transcranial magnetic stimulation-evoked potentials that can be monitored with electroencephalography. Effective connectivity refers to causal connections in the brain; it describes how different brain areas communicate with each other. Broca's area is crucial for all phases of speech processing and is located in the frontotemporal region of the cortex. Only a few studies have investigated this region using transcranial magnetic stimulation-electroencephalography because of the large cranial muscles that are located over these areas, resulting in large artifacts covering the transcranial magnetic stimulation-evoked potentials. However, it is shown that this obstacle can be overcome with new artifact-removal tools. We used minimum-norm estimation to locate the sources of the neuronal signals in electroencephalography data after stimulating the right-hemispheric homologue of Broca's area in three right-handed subjects; it was shown that the spreading of brain activity might be different for different individuals and that the brain activity spread fast to the contralateral hemisphere.

The effect of experimental pain on short-interval intracortical inhibition with multi-locus transcranial magnetic stimulation.

Chronic neuropathic pain is known to alter the primary motor cortex (M1) function. Less is known about the normal, physiological effects of experimental neurogenic pain on M1. The objective of this study is to determine how short-interval intracortical inhibition (SICI) is altered in the M1 representation area of a muscle exposed to experimental pain compared to SICI of another muscle not exposed to pain. The cortical representation areas of the right abductor pollicis brevis (APB) and biceps brachii (BB) muscles of 11 subjects were stimulated with a multi-locus transcranial magnetic stimulation device while the resulting motor-evoked potentials (MEPs) were recorded with electromyography. Single- and paired-pulse TMS was administered in seven conditions, including one with the right hand placed in cold water. The stimulation intensity for the conditioning pulses in the paired-pulse examination was 80% of the resting motor threshold (RMT) of the stimulated site and 120% of RMT for both the test and single pulses. The paired-pulse MEP amplitudes were normalized with the mean amplitude of the single-pulse MEPs of the same condition and muscle. SICI was compared between conditions. After the cold pain, the normalized paired-pulse MEP amplitudes decreased in APB, but not in BB, indicating that SICI was potentially increased only in the cortical area of the muscle subjected to pain. These data suggest that SICI is increased in the M1 representation area of a hand muscle shortly after exposure to pain has ended, which implies that short-lasting pain can alter the inhibitory balance in M1.

Individual Activation Patterns After the Stimulation of Different Motor Areas: A Transcranial Magnetic Stimulation-Electroencephalography Study.

The combination of transcranial magnetic stimulation (TMS) and electroencephalography (EEG) enables one to study effective connectivity and activation order in neuronal networks. To characterize effective connectivity originating from the primary motor cortex (M1), dorsal premotor area (PMd), and supplementary motor area (SMA). Three right-handed volunteers (two men, aged 25-30 years) participated in a navigated TMS-EEG experiment. M1, PMd, and SMA over the nondominant hemisphere were stimulated with 150 TMS pulses. Minimum-norm estimates were derived from the EEG data to estimate the spatial spreading of TMS-elicited neuronal activation on an individual level. The activation order of the cortical areas varied depending on the stimulated area. There were similarities and differences in the spatial distribution of the TMS-evoked potentials between subjects. Similarities in cortical activation patterns were seen at short poststimulus latencies and the differences at long latencies. This pilot study suggests that cortical activation patterns and the activation order of motor areas differ interindividually and depend on the stimulated motor area. It further indicates that TMS-activated effective connections or underlying structural connections vary between subjects. The spatial patterns of TMS-evoked potentials differ between subjects especially at long latencies, when probably more complex neuronal networks are active.

[Surgical treatment for clinical stage II and III small cell lung cancer patients].

To evaluate a surgical resection in multidisciplinary treatment for clinical stage (c-stage) II and III small cell lung cancer (SCLC) patients, we examined the postoperative prognosis concerning in 18 patients who underwent complete surgical resection. Five-year survival rate of 8 c-stage II and 10 c-stage III patients showed 62.5% and 46.7%, respectively. Of these, 7 patients underwent preoperative induction chemotherapy resulting in down-staging to c-stage 0-I, and their survival rate at 5 years after sugery was as good as 68.6%. However, in 4 (57.1%) of 7 patients, tumor cells proved to be alive by pathological assessment of lymph nodes. As a consequence, it was suggested that surgery for c-stage II and III SCLC patients is a meaningful treatment considering their prognoses. In particular, even though the patients show a favorable response of preoperative chemotherapy, to completely eliminate remnant tumor cells, a surgical resection may be necessary.

Rapid and accurate identification of microorganisms contaminating cosmetic products based on DNA sequence homology.

The aim of this study was to develop rapid and accurate procedures to identify microorganisms contaminating cosmetic products, based on the identity of the nucleotide sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA coding DNA (rDNA). Five types of microorganisms were isolated from the inner portion of lotion bottle caps, skin care lotions, and cleansing gels. The rDNA ITS region of microorganisms was amplified through the use of colony-direct PCR or ordinal PCR using DNA extracts as templates. The nucleotide sequences of the amplified DNA were determined and subjected to homology search of a publicly available DNA database. Thereby, we obtained DNA sequences possessing high similarity with the query sequences from the databases of all the five organisms analyzed. The traditional identification procedure requires expert skills, and a time period of approximately 1 month to identify the microorganisms. On the contrary, 3-7 days were sufficient to complete all the procedures employed in the current method, including isolation and cultivation of organisms, DNA sequencing, and the database homology search. Moreover, it was possible to develop the skills necessary to perform the molecular techniques required for the identification procedures within 1 week. Consequently, the current method is useful for rapid and accurate identification of microorganisms, contaminating cosmetics.

[Prevention of parenchymal air leakage after lung resection; comparison of effectiveness in drug formation of fibrin adhesive].

We investigated the comparative effectiveness of the seat and glue types of fibrin adhesive, to clarify which is more useful in preventing postoperative parenchymal air leaks after lung resection. One hundred sixteen patients received fibrin adhesive to prevent postoperative air leakage after lung resection carried out by the same surgeon. Ninety-two lobectomies and 24 partial resections were assessed. There were 29 patients with emphysematous lung. In the seat type group, an average of 2.6 postoperative days elapsed before chest drain removal. In the glue type, this average was 3.2 days, a significant difference. This difference was also evident in the lobectomy group. However, among emphysematous lung patients and the partial resection group, there was no apparent difference. Prolonged air leakage was seen in 2 patients with emphysematous lung, but no difference in terms of drug formation could be seen. The seated type of fibrin adhesive was more useful than the glue type in preventing postoperative air leaks, but in emphysematous lung patients, better surgical technique would seem to be the critical factor.

Sequence of the Clostridium thermocellum mannanase gene man26B and characterization of the translated product.

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.

A cryptic plasmid, pAO1, from a compost bacterium, Bacillus sp.

The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed. Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus. The plasmid pAO1 was 3,325 bp in size. Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence. The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans. A putative origin sequence for a plus-strand was located between ORFs. Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis.

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.

Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21.

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.

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli.

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.

Sequence of celQ and properties of celQ, a component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids. The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain. Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only. CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan. The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC. By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme. Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C. thermocellum cellulosome.

Recognition of receptor lipopolysaccharides by spike G protein of bacteriophage phiX174.

The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.

A large gene cluster for the Clostridium cellulovorans cellulosome.

A large gene cluster for the Clostridium cellulovorans cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39-47, 1998). Gene walking revealed that the engL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244-247, 2000) was located downstream of the cbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes is cbpA-exgS-engH-engK-hbpA-engL-man A-engM-engN and is about 22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene. This large gene cluster is flanked at the 5' end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regA and at the 3' end by noncellulosomal genes with homology to transposase (trp) and malate permease (mle). Since gene clusters for the cellulosome are also found in C. cellulolyticum and C. josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.

Purification, characterization, and molecular cloning of acidophilic xylanase from penicillium sp.40.

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.

Characterization of the cellulolytic complex (cellulosome) from Ruminococcus albus.

The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albus strain 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 x 10(6) Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40kDa 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-Doc-VII 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. albus resembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellum and 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.