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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.

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.

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 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.

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 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.

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.

To identify chemicals with endocrine-disrupting activity easily, we developed a new bioassay system, consisting of bioassay using genetically modified yeast expressing human estrogen receptor and high performance liquid chromatography (HPLC), in which advantages of instrumental analysis and bioassay are combined. The peaks in the mixture of these estrogen-like compounds analyzed using an HPLC bioassay were similar to those obtained by analysis using an HPLC-UV detector. Underground water and sea sediment were analyzed by an HPLC bioassay, and detected a few estrogen-like compounds, respectively. Estrogen-like compounds and yeast-growth inhibitors can be separated by HPLC-bioassay.

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.

The porphobilinogen deaminase encoded by the Clostridium josui hemC gene was purified from a recombinant Escherichia coli strain and its properties were characterized. The optimal temperature and pH of the purified enzyme were 65 degrees C and 7.0, respectively. This enzyme was quite thermostable: it retained 86% of the original activity after incubation at 70 degrees C for 1 h. The Km and Vmax values of the enzyme were 65 microM and 3.3 micromol/h/mg for porphobilinogen, respectively.

A multienzyme complex, cellulosome, of the facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6 was produced on microcrystalline cellulose (Avicel) under aerobic conditions. During growth on Avicel, the bacterial cells were found to be capable of adhesion to Avicel by scanning electron microscopic (SEM) analysis. The multienzyme complex of P. curdlanolyticus B-6 was isolated from the crude enzyme preparation by gel filtration chromatography on Sephacryl S-300 and affinity purification on cellulose. The isolated multienzyme complex was able to bind to both Avicel and insoluble xylan and consists of cellulolytic and xylanolytic enzymes such as avicelase, carboxymethyl cellulase (CMCase), cellobiohydrolase, β-glucosidase, xylanase, β-xylosidase and α- l -arabinofuranosidase. The molecular mass of the complex was estimated to be 1600 kDa. It composed of at least 12 proteins on SDS-PAGE and 10 CMCases and 11 xylanases on zymograms. The isolated multienzyme complex could degrade the raw lignocellulosic substances effectively.

In Materials and Methods, page 401, replace the final two lines of the left columnwith the following: “For CLSM observation, the biofilm sample was fixed with glutaraldehyde, stained with 200 mL of 0.9 mg/mL GFP-CBM3 and 200 mL of 10 mM SYTO 60 Red Fluorescent Nucleic Acid Stain (Molecular Probes, OR, USA), and incubated for 15 min in the dark”. On page 401, replace lines 3e5 of the right columnwith the following: “In separate experiments, the sample was stained with 200 mL of 90 mg/mL wheat germ agglutinin-Oregon 488 conjugate (WGA) (Molecular Probes) (10) and SYTO 60 dye”.

A beta-N-acetylglucosaminidase gene (nag84A) was cloned from Clostridium paraputrificum M-21 in Escherichia coli. The nag84A gene consists of an open reading frame of 4647 by encoding 1549 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-acetylchitohexaose, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta-D-glucosaminide [4-MU-(G1cNAc)], but did not hydrolyze 4-MU-beta-D-glucoside, 4-MU-alpha-D-glycoside, 4-MU-alpha-D-GlcNAc, 4-MU-alpha-D-galactoside, 4-MU-beta-D-xyloside, PNP-beta-D-galactoside, and PNP-alpha-D-xyloside. The enzyme was optimally active at 50 degrees C and pH 6.5, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 8.5 microM and 1.39 micromol/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 beta-N-acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family 18.

Clostridium paraputrificum M-21 was transformed with several shuttle plasmids constructed for Clostridium acetobutylicum-Escherichia coli and Clostridium perfringens-E. coli by electroporation. The Clostridium stercorarium xylanase gene xyn10B was successfully expressed in C. paraputrificum M-21 and the expressed protein did not suffer from proteolysis by host protease(s). This system will provide us with a genetic tool for genetic and metabolic engineering of this bacterium.

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

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-beta-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-beta-D-xylobiosides as substrates. The k(cat) values determined using o-nitrophenyl-beta-D-xylobioside did not change as the pH increased, whereas the k(cat) values obtained with BWX, phenyl-beta-D-xylobioside and p-nitrophenyl-beta-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-beta-D-xylobiosides in the characterization of xylanases.

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.

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 H 2 /mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°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 .

Chitinase B (ChiB) was purified from the culture supernatant of Xanthomonas sp. strain AK by Phenyl-Toyopearl 650M and DEAE-Toyopearl 650M column chromatographies. The purified enzyme preparation gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of ChiB was estimated to be 48,000. The enzyme was optimally active at pH 6.0 and 60°C. N-Terminal amino acid sequence analysis suggested that ChiB is a member of glycosyl hydrolase family 18 and that it is genetically different from ChiA recently reported (Sakka et al., J. Ferment. Bioeng., 86, 527–533, 1998). Immunological analysis suggested that ChiB was the major chitinase species in the culture supernatant of Xanthomonas sp. strain AK and that production of the enzyme was induced by the presence of chitin.