Molecular breeding of transgenic rice expressing a xylanase domain of the xynA gene from Clostridium thermocellum.

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 novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose.

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.

Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.

The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548-554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan. The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

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.

The multidomain xylanase Xyn10B as a cellulose-binding protein in Clostridium stercorarium.

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.

Sequence analysis and overexpression of a pectin lyase gene (pel1) from Aspergillus oryzae KBN616.

A gene (pel1) encoding pectin lyase (Pel1) was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,196 bp with a single intron. The ORF encoded 381 amino acids with a signal peptide of 20 amino acids. The deduced amino acid sequence showed high similarity to those of Aspergillus niger pectin lyases and Glomerella cingulata PnlA. The pel1 gene was successfully overexpressed under the promoter of the A. oryzae TEF1 gene. The molecular mass of the recombinant pectin lyase substantially coincided with that calculated based on nucleotide sequence.

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.

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products.

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.

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.

Characterization of N-acetylmuramidase M-1 of Streptomyces globisporus produced by Escherichia coli BL21(DE3)pLysS.

A gene of Streptomyces globisporus encoding N-acetylmuramidase M-1 was cloned into the pET26b vector and expressed in Escherichia coli BL21(DE3)pLysS. Maximal activity for the purified enzyme was observed at 55 degrees C with an optimal pH of 5.3, and N-bromosuccinimide strongly inhibited lytic activity even at a concentration of 0.01 mM. The enzyme showed N,O-diacetylmuramidase activity.

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.

Isolated cardiac metastasis from sacral chordoma.

A 64-year-old woman presented with right heart failure caused by a cardiac tumor centered in the free wall of the right ventricle, accompanied by pericardial effusion. A match between the biopsy specimen and tissue removed 4 years earlier resulted in the diagnosis of a cardiac metastasis from a chordoma. Immunohistochemical staining was also useful in establishing the diagnosis. To alleviate the right ventricular outflow obstruction, a palliative operation was planned, resecting the tumor and performing a right ventriculoplasty, which was cancelled due to the extent of infiltration of the tumor, and instead a right atrium to pulmonary artery shunt was attempted using a vascular prosthesis, only to fail due to an inability to maintain blood flow through the prosthesis. Presently there are no definitive treatment options available, and some palliative chemotherapy is being performed. Single cardiac metastases from a chordoma are extremely rare.

Influence of angiotensin-converting enzyme gene polymorphism on development of athlete's heart.

BACKGROUND AND HYPOTHESIS: Genetic influence on development of athlete's heart is uncertain. This study investigated whether angiotensin-converting enzyme (ACE) gene polymorphism influenced development of athlete's heart. METHODS: Forty-three participants in a 100-km ultramarathon were classified on the basis of ACE gene polymorphism into a deletion group (n = 26) and an insertion group (n = 17). Echocardiograms were recorded to determine left ventricular end-diastolic and end-systolic diameters, interventricular septal thickness, left ventricular posterior wall thickness, left ventricular mass, and ejection fraction. RESULTS: Left ventricular end-diastolic diameter (65.5 +/- 4.0 mm) and left ventricular mass (369.5 +/- 73.9 g) were significantly larger in the subjects with deletion than in those with insertion (57.4 +/- 4.2 mm, 306.5 +/- 93.7 g). However, no significant differences in the other parameters were noted. CONCLUSIONS: In long-distance runners, ACE gene polymorphism of the D/D and D/I genotypes has a stronger influence on left ventricular hypertrophy than polymorphism of the I/I genotype.

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.

Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain.

The Ruminococcus albus F-40 egV gene, 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. albus F-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 recombinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(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 degrees C, respectively. By Western blot analysis using the antiserum raised against the truncated enzyme, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium.

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl-D-glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H2/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45 degrees C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.

Molecular breeding of cellulolytic microbes, plants, and animals for biomass utilization.

Cellulosic materials are the major components of fibrous biomass produced as a result of photosynthesis and are considered as a reservoir of solar energy and organic materials. In order to cope with the problems of food and energy shortages expected in the near future, biotechnologists are encouraged to develop new technologies for the more effective utilization of the world's sustainable resources, i.e., biomass. One way is to engineer microorganisms and animals with the capability of digesting and utilizing cellulosic materials, and plants which can be easily degraded by cellulolytic enzymes. In this article, we summarize recent studies on the molecular breeding of cellulolytic organisms for biomass utilization along with some considerations regarding cellulolytic enzymes.

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product.

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.