Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Enhancing yield of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by controlling NH<Subscript>4</Subscript><Superscript>+</Superscript> concentration

Yield of S-adenosylmethionine was improved significantly in recombinant Pichia pastoris by controlling NH₄ ⁺ concentration. The highest production rate was 0.248 g/L h when NH₄ ⁺ concentration was 450 mmol/L and no repression of cell growth was observed. Within very short induction time (47 h), 11.63 g/L SAM was obtained in a 3.7 L bioreactor.     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.     

Protective Effect of Enzymatic Extracts from Microalgae Against DNA Damage Induced by H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The enzymatic extracts from seven species of microalgae (Pediastrum duplex, Dactylococcopsis fascicularis, Halochlorococcum porphyrae, Oltmannsiellopsis unicellularis, Achnanthes longipes, Navicula sp. and Amphora coffeaeformis) collected from three habitats (freshwater, tidal pool, and coastal benthic) at Jeju Island in Korea were investigated for their antioxidant activity. Of the extracts tested, the AMG 300 L (an exo 1, 4-α-d-glucosidase) extract of P. duplex, the Viscozyme extract of Navicula sp., and the Celluclast extract of A. longipes provided the most potential as antioxidants. Meanwhile, the Termamyl extract of P. duplex in an H₂O₂ scavenging assay exhibited an approximate 60% scavenging effect. In this study, we report that the DNA damage inhibitory effects of P. duplex (Termamyl extract) and D. fascicularis (Kojizyme extract) were nearly 80% and 69% respectively at a concentration of 100 μg/ml. Thus, it is suggested that the microalgae tested in this study yield promising DNA damage inhibitory properties on mouse lymphoma L 5178 cells that are treated with H₂O₂. Therefore, microalgae such as P. duplex may be an excellent source of naturally occurring antioxidant compounds with potent DNA damage inhibition potential.     

Effects of increasing concentrations of sodium metabisulphite (Na<Subscript>2</Subscript>S<Subscript>2</Subscript>O<Subscript>5</Subscript>, SBS) on deoxynivalenol (DON) concentration and microbial spoilage of triticale kernels preserved without and with propionic acid at various moisture contents

Unground triticale kernels contaminated with 6.63 mg deoxynivalenol (DON) per kg dry matter were stored for up to 63 days at total moisture contents of 13 and 15% in order to study the time-dependent kinetics of DON concentration in dependence on graded levels of sodium metabisulfite [0, 1, 2, 3, 4 and 5 g Na₂S₂O₅ (SBS) per kg], and in the absence and presence of 10 g propionic acid (PA) per kg. The DON concentration decreased with increasing amounts of supplemented SBS and with increasing duration of the preservation period in a bi-exponential fashion when SBS addition was ≥3 g/kg. Lower SBS concentrations yielded inconsistent results. The maximum measured DON reductions after adding 5 g SBS/kg were 3 and 4% of the initial DON concentration after 63 days in the absence and presence of PA at moisture contents of 15%, while the corresponding recovery for the variants preserved at 13% amounted to 21 and 11%, respectively. The 12 variants preserved without PA supplementation were more frequently contaminated by moulds and yeasts (n = 5) than the corresponding variants stored together with PA (n = 1). The overall results and regressive evaluations do suggest that the highest SBS addition of 5 g/kg triticale at a moisture content of 15% preserved for 63 days would be necessary for a maximum DON reduction. Although PA did not exert a direct decontaminating effect, an additional supplementation together with SBS seemed to be advantageous with regard to the prevention of yeast and mould contamination and favouring the decontamination reaction by the acid milieu.     

Production of bioethanol from sugarcane bagasse using NH<Subscript>4</Subscript>OH-H<Subscript>2</Subscript>O<Subscript>2</Subscript> pretreatment and simultaneous saccharification and co-fermentation

In this study, we investigated the production of bioethanol from sugarcane bagasse (SCB) using an NH₄OH-H₂O₂ pretreatment and simultaneous saccharification and co-fermentation (SScF). Response surface methodology and a 2³ Box-Behnken design were used to evaluate the effect of different liquid mixture concentrations, liquid-to-solid ratios (LSRs) and pretreatment temperatures on the production of ethanol. The liquid mixture concentration and LSR significantly influenced the fermentation efficiency. Based on ridge max analysis, the following pretreatment conditions resulted in a fermentation efficiency of 95.79 ± 0.01%: liquid mixture concentration 53%, LSR 28, and a temperature of 63°C. A morphological analysis performed using scanning electron microscopy (SEM) and chemical characterization revealed that these pretreatment conditions were effective in disrupting the sugarcane fibers and removing lignin. Ethanol fermentation with the pretreated SCB using SScF in yeast SHY 07-1 resulted in an ethanol concentration of 14.65 ± 0.17 g/L, an ethanol yield of 0.48 ± 0.01 g/g, and an ethanol productivity of 0.12 ± 0.01 g/(L/h), which represents increases of 106.02, 89.98, and 107.02%, respectively, over the values obtained from SScF with untreated SCB.     

Interindividual differences in initial DNA repair capacity when evaluating H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.     

C<Subscript>4</Subscript> plants in the deserts of China: occurrence of C<Subscript>4</Subscript> photosynthesis and its morphological functional types

C₄ photosynthetic pathway and morphological functional types were determined for 104 species in 45 genera and 10 families from the deserts of China. 67 C₄ species (64.4 %) were found in Dicotyledoneae (e.g. Chenopodiaceae, Polygonaceae, and Amaranthaceae), the other 37 species were in Monocotyledoneae (e.g. Gramineae, Cyperaceae, and Commelinaceae). 36.5 % of the Chenopodiaceae species (predominantly members of the genera Anabasis, Atriplex, Kochia, Salsola, and Suaeda) identified in the desert regions were found with C₄ photosynthesis, which was about 48 % of the total C₄ species. Many C₄ species (58.7 %) were annuals (e.g. Amaranthus, Atriplex, Digitaria, Eragrostis, Kochia, and Salsola) and experienced long-term droughts, high temperature, and high irradiance. Relatively more shrub C₄ species (28 species of 104) were found in Chenopodiaceae (e.g. Anabasis, Camphorosma, Haloxylon, and Salsola) and Polygonaceae (e.g. Calligonum) in the desert regions. Most of shrub C₄ species with small leaf area were no more than 1 m in height and distributed in sandy soils. Composition of relatively more annual species, shrubs, and Chenopodiaceae C₄ species was the primary characteristic for the C₄ species occurrence in deserts, and this was remarkably related with the arid environmental conditions.     

Activity of C<Subscript>4</Subscript> enzymes in C<Subscript>3</Subscript>-type herbaceous plants

The activity of enzymes characteristic for C₄-type photosynthesis was determined in different organs of two herbaceous plants: Reynoutria japonica Houtt. and Helianthus tuberosus L. The activity of phosphoenolpyruvate carboxylase (PEPC) was usually higher in the roots, some of the stem tissues and petioles in comparison to the leaf blades. The highest activity of malic enzymes (NAD-ME, NADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK) was in the petioles and stem tissues of both plants and the lowest in the leaf blades and the pith of Helianthus tuberosus L.     

Effects of elevated atmospheric CO<Subscript>2</Subscript> on the nutritional ecology of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> grass-feeding caterpillars

It is plausible that the nutritional quality of C₃ plants will decline more under elevated atmospheric CO₂ than will the nutritional quality of C₄ plants, causing herbivorous insects to increase their feeding on C₃ plants relative to C₄ plants. We tested this hypothesis with a C₃ and C₄ grass and two caterpillar species with different diet breadths. Lolium multiflorum (C₃) and Bouteloua curtipendula (C₄) were grown in outdoor open top chambers at ambient (370 ppm) or elevated (740 ppm) CO₂. Bioassays compared the performance and digestive efficiencies of Pseudaletia unipuncta (a grass-specialist noctuid) and Spodoptera frugiperda (a generalist noctuid). As expected, the nutritional quality of L. multiflorum changed to a greater extent than did that of B. curtipendula when grown in elevated CO₂; levels of protein (considered growth limiting) declined in the C₃ grass, while levels of carbohydrates (sugar, starch and fructan) increased. However, neither insect species increased its feeding rate on the C₃ grass to compensate for its lower nutritional quality when grown in an elevated CO₂ atmosphere. Consumption rates of P. unipuncta and S. frugiperda were higher on the C₃ grass than the C₄ grass, the opposite of the result expected for a compensatory response to the lower nutritional quality of the C₄ grass. Although our results do not support the hypothesis that grass-specialist insects compensate for lower nutritional quality by increasing their consumption rates more than do generalist insects, the performance of the specialist was greater than that of the generalist on each grass species and at both CO₂ levels. Mechanisms other than compensatory feeding, such as increased nutrient assimilation efficiency, appear to determine the relative performance of these herbivores. Our results also provide further evidence against the hypothesis that C₄ grasses would be avoided by insect herbivores because a large fraction of their nutrients is unavailable to herbivores. Instead, our results are consistent with the hypothesis that C₄ grasses are poorer host plants primarily because of their lower nutrient levels, higher fiber levels, and greater toughness.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

Components of CO<Subscript>2</Subscript> exchange in leaves of C<Subscript>3</Subscript> species with different ability of starch accumulation

Using a radiogasometric method the rates of photorespiratory and respiratory decarboxylations of primary and stored photosynthates in the leaves of two groups of C₃ species, differing in the ability of starch accumulation, were determined. One group included starch-accumulating (SA) species with rates of starch synthesis on the average 38 % the rate of photosynthesis [Solanum tuberosum L., Arabidopsis thaliana (L.) Heynh, Helianthus annuus L., and Plantago lanceolata L.]. The second group represented starch-deficient (SD) species with rates of starch synthesis less than 8 % the rate of photosynthesis (Secale cereale L., Triticum aestivum L., Hordeum vulgare L., and Poa trivialis L.). In SA species the rate of respiration in the dark was significantly higher than in SD species. No differences were found in the rates of photosynthesis, photorespiration, and respiration under irradiation. Thus, the degree of inhibition of respiration by irradiation was in SA species higher than in SD species. It is concluded that starch does not provide substrates for respiratory and photorespiratory decarboxylations in irradiated photosynthesizing leaves.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Formation of extracellular polymeric substances from acidogenic sludge in H<Subscript>2</Subscript>-producing process

In this study, the formation of extracellular polymeric substances (EPS) and surface characteristics of an acidogenic sludge in anaerobic H₂-producing process was investigated. Results show that carbohydrates, proteins, and humic substances were the dominant components in bound EPS (BEPS), while in soluble EPS (SEPS), carbohydrates were the main component. The total content of BEPS initially increased but then kept almost unchanged during fermentation from 25 to 35 h; after that, it slightly decreased. The total content of SEPS increased to 172.5 ± 0.05 mg C g⁻¹ volatile suspended solid with the time that increased to 23.5 h, and then rapidly decreased until 43 h; thereafter, it kept almost unchanged. The SEPS had good correlations with the specific H₂ production rate, substrate degradation rate, and specific aqueous products formation rate, but the BEPS seemed to have no such correlations with these specific rates. Results also confirm that part of EPS could be utilized by the H₂-producing sludge. As the substrate was in short supply, the EPS would be hydrolyzed to sever as carbon and energy source.     

Cell growth and P(3HB) accumulation from CO<Subscript>2</Subscript> of a carbon monoxide-tolerant hydrogen-oxidizing bacterium, <Emphasis Type="Italic">Ideonella</Emphasis> sp. O-1

Cell growth and accumulation of polyhydroxybutyric acid, P(3HB), from CO₂ in autotrophic condition of a newly isolated hydrogen-oxidizing bacterium, the strain O-1, was investigated. The bacterium, which was deposited in the Japan Collection of Microorganisms as JCM17105, autotrophically grows by assimilating H₂, O₂, and CO₂ as substrate. 16S rRNA gene sequence of the bacterium was the closest to Ideonella dechloratans (99%). Specific growth rate of the strain O-1 was faster than a hydrogen-oxidizing bacterium, Ralstonia eutropha, which is well-known P(3HB)-producing microorganism. The strain O-1 is tolerant to high O₂ concentration and it can grow above 30% (v/v) O₂, while the growth of R. eutropha and Alcaligenes latus was seriously inhibited. In culture medium containing 1 g/L (NH₄)₂SO₄, cell concentration of the strain O-1 and P(3HB) increased to 6.75 and 5.26 g/L, respectively. The content of P(3HB) in the cells was 77.9% (w/w). The strain O-1 was very tolerant to carbon monoxide (CO) and it grew even at 70% (v/v) CO, while the growth of R. eutropha and A. latus were seriously inhibited at 5% (v/v) CO. From these results, it is expected that the strain O-1 will be useful in the manufacture of P(3HB) because the industrial exhaust gas containing CO₂, H₂, and CO can be directly used as the substrate in the fermentation process.     

Molecular modeling and dynamics simulation of a histidine-tagged cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript>

Although an affinity tag such as six consecutive histidines, (His)₆-tag, has been widely used to obtain high quantity of recombinant proteins, little is known about its influences on heme proteins for lack of structural information. When (His)₆-tag was introduced to the N-terminus of a small heme protein, cytochrome b ₅, experimental results showed the resultant protein, (His)₆-cyt b ₅, has similar property and function to that of isolated cyt b ₅. To provide structural information for this observation, we herein performed a structural prediction of (His)₆-cyt b ₅ by molecular modeling in combination with molecular dynamics simulation. The predicted structure, as assessed by a series of criteria with good quality, reveals that the (His)₆-tag adopts a helical conformation and packs against the hydrophobic core 2 of cyt b ₅ through salt bridges, hydrogen bonding and hydrophobic interactions. The heme group, with the axial His ligands slightly rotated, was found to have similar conformation as in isolated cyt b ₅, which indicates that the N-terminal (His)₆-tag does not alter the heme active site, resulting in similar dynamics properties for core 1. This study provides valuable information of interactions between (His)₆-tag and the rest of the protein, aiding in rational design and application of functional His-tagged proteins.     

Stress-protective activity of the CH<Subscript>3</Subscript>CO-Lys-Lys-Arg-Arg-NH<Subscript>2</Subscript> synthetic peptide (protectin)

The CH₃CO-Lys-Lys-Arg-Arg-NH₂ peptide (the author has named it protectin) was synthesized, and its activity was studied during different stress actions. Protectin was found to normalize the content of corticosterone and adrenalin in adrenal glands and blood after its intranasal administration to rats one day before a cold or heat shock, or hypobaric hypoxia at doses of 1–10 μg/animal and after its intravenous administration just after acute hemorrhage at doses of 0.5–2 μg/animal. The intranasal administration of protectin at doses of 1–10 μg/rat one day before the heat or cold shock was also shown to prevent a change in the content of free histamine and the activity of diamine oxidase in myocardium, which was induced by the dramatic change in the activity of the enzyme after the temperature actions.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.