Yuwadee Peerapornpisal

Purification and Characterization of Membrane-bound Hydrogenase from Hydrogenobacter thermophilus Strain TK-6, an Obligately Autotrophic, Thermophilic, Hydrogen-oxidizing Bacterium

A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with α2β2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 μM and with a turnover number around 2900 (min-1). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.

Reversed-phase liquid chromatographic–mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions

Reversed-phase HPLC coupled to the atmospheric pressure ionization-electrospray ionization (API-ESI) MS was used for microcystin-LR detection and quantitation in samples of dried Microcystis aeruginosa cells. An alkaline linear gradient (20 mmol/l ammonium hydroxide-acetonitrile, pH 9.7) was used for elution of the toxic peptides. Limit of detection was 1 microg/ml (20 ng per injection) in the scan mode of MS and 0.1 microg/ml (2 ng per injection) in the case of selective ion monitoring.

Thermostablility of phycobiliproteins and antioxidant activity from four thermotolerant cyanobacteria

Four cyanobacterial strains including Cyanosarcina sp. SK40, Phormidium sp. PD40‐1, Scytonema sp. TP40 and Leptolyngbya sp. KC45 were selected and investigated for the phycobiliprotein (PBP) content and thermostable antioxidant activity of their cell‐free extracts. The highest content of 181.63 mg/g dry weight phycobiliprotein was found in Leptolyngbya sp. KC45 with phycoerythrin (PE) as the main phycobiliprotein. Among the PBPs of four thermotolerant cyanobacteria, PE from Leptolyngbya sp. KC45 exhibited the highest thermal stability as 80% of the original level remained after being heated at 60°C for 30 min. Antioxidant activities were detected in the cell‐free extracts of all cyanobacteria and that of Leptolyngbya sp. KC45 was also found in the highest value of 7.44 ± 0.14 and 3.89 ± 0.08 mg gallic acid equivalent (GAE) g−1 dry weights determined by 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH) and reducing power assay, respectively. This also corresponded to the phenolic compound content. Based on DPPH and reducing power assay, antioxidant activities of all cyanobacterial extracts showed the high thermostability as approximately 80% remained after being heated at 80°C for 30 min. However, it clearly indicated that the thermostability of antioxidant activity from the hot spring cyanobacterial cell‐free extract was not contributed only by the PE, but also came from phenolic compounds and other oxidative substances.

Isolation and cultivation of thermophilic cyanobacteria from hot springs of northern Thailand

Abstract Three cyanobacterial strains were obtained from hot springs of Northern Thailand. Morphological and physiological studies revealed that all three strains belong to genus Chroococcidiopsis. One of these strains, TS-821, was used for further studies because of its stable autotrophic growth. Its optimum temperature for growth is around 50°C, and its optimum pH for growth is around 8. The strain is capable of dinitrogen fixation, and is tolerant against nitrite (5 mM), sulfite (0.6 mM) and a high concentration of carbon dioxide in the gas phase (0.4 atm). These properties indicate that the strain can be used for the conversion to organic matter of carbon dioxide produced by combustion.

Application of desmid diversity in assessing the water quality of 12 freshwater resources in Thailand

Desmids from 12 freshwater resources in the northern part of Thailand were investigated during 2002 to 2003. A total of 91 taxa were found. They belonged to 17 genera: Actinotaenium, Spirotaenia, Netrium, Gonatozygon, Pleurotaenium, Closterium, Euastrum, Micrasterias, Cosmarium, Cosmocladium, Stuarastrum, Staurodesmus, Xanthidium, Teilingia, Spondylosium, Hyalotheca and Desmidium. The water qualities in all the water resources were classified as oligotrophic to meso-eutrophic by trophic status. The taxa that could possibly be used as bioindicators of trophic state were Staurastrum gutwinskii, Spondylosium pandurifoemae, Cosmarium capitulum, C. mediosrobiculatum var. egranutum, S. tortum, Closterium gracile var. elongatum, C. kuetzingii and Closterium dianae var. dianae. The most frequently found taxa were Staurastrum limneticum var. burmense, S. tetracerum var. tetraerum, Pleurotaenium trabecula, Closterium ehrenbergii var. ehrenbergii and C. kuetzingii. The rare taxa in this study were Actinotaenium sp. Spirotaenia condensata, Pleurotaenium burmense var. dacchense and Micrasterias apiculata. Forty-one taxa of desmids were identified as new records for Thailand.

Growth characteristics and dense culture of a thermophilic cyanobacterium, Chroococcidiopsis sp. strain TS-821

Abstract A thermophilic cyanobacterium, Chroococcidiopsis sp. strain TS-821, was cultivated under illumination at various intensities of and at various concentrations of medium components in a 1- l photobioreactor. The specific growth rate and the dry cell weight at the end of cultivation were determined. On the basis of the results obtained, cultivation under the optimum conditions was carried out for achieving a dense culture. Cultivation was also done under conditions of CO 2 supply in two different modes: (i) 10% CO 2 throughout, and (ii) 10% at the beginning and then 20% at the termination of linear growth. The dry cell yield per liter at the end of the culture was 70% higher than that in the case of standard culture. The total sugar content of the cells reached 30% of the dry cell weight at the end of the dense culture, which is higher than that of other cyanobacterium, Synechococcus elongatus PCC 6301. The supernatant of a buffer suspension of cells which had been collected at the end of the dense culture and freeze-dried was clear blue. The absorption spectrum of this supernatant was measured and compared with those of the cell extract and of a phycobiliprotein extracted from cell of this strain. The wavelengths of two absorption maxima from the supernatant coincided with those from the phycobiliprotein. The results show that the phycobiliprotein can easily be excreted from the cells. The results of polyacrylamide gel electrophoresis and spectrophotometrical analyses suggest that the excreted phycobiliprotein is a phycocyanin. Furthermore, when total protein, lipid, and sugar contents were measured without removal of the culture medium, the total sugar content was about 50% of the cell component. This indicates that the sugar was excreted into the medium.

Effect of carbon dioxide concentration on the growth and RubisCO activity of a thermophilic cyanobacterium, Chroococcidiopsis sp. strain TS-821

Abstract A thermophilic cyanobacterium, Chroococcidiopsis sp. strain TS-821 was cultivated under various CO2 concentrations using 1-l photobioreactor. The strain can grow well under aeration without addition of any extra CO2. Addition of 5 or 10% CO2 in the flow gas did not show significant effects on the growth of the strain, but aeration with a gas mixture containing 15 or 20% CO2 inhibited the growth. RubisCO activity in the cells showed higher values when cultivated under 5 or 10% CO2 than when cultivated under air, but those values under 15 or 20% CO2 were very low. Little difference was observed in the final amount of RubisCO protein in the cells cultivated under different CO2 concentration conditions. This indicated that RubisCO activity was controlled not transcriptionally but post-translationally by change in CO2 concentration during cultivation.

Cancer Chemopreventive Effect of Spirogyra Neglecta (Hassall) Kützing on Diethylnitrosamine-Induced Hepatocarcinogenesis in Rats

Spirogyra neglecta, a freshwater green alga, is a local food in the northern and northeastern parts of Thailand. This investigation explored the anticarcinogenicity of S neglecta and its possible cancer chemopreventive mechanisms in rats divided into 14 groups. Groups 1 and 10 served as positive and negative control groups, respectively. Groups 1-9 were intraperitoneally injected with diethylnitrosamine (DEN) once a week for 3 weeks. Groups 10-14 received normal saline instead. One week after the last DEN injection, groups 2-5 were administered for 9 consecutive weeks various doses of S neglecta extract (SNE) and dried S neglecta (SND), mixed with basal diet. Groups 6-9 and 11-14 similarly were administered various doses of SNE and SND starting from the first week of the experiment. Administration of SNE and SND was not associated with formation of glutathione-S- transferase placental form (GST-P) positive foci in rat liver. SNE and SND during initiation phase significantly reduced the number of GST-P positive foci in rats injected with DEN. The number of GST-P also diminished in groups treated with SNE and SND after injection with DEN, except for the low dose extract group. SNE showed stronger anticarcinogenic potency than SND. Furthermore, SNE also decreased the number of Ki-67 positive cells. However, the numbers of TUNEL-positive cells in the liver of the SNE-treated groups were not statistically different from the controls. The GST activity in 50 mg/kg bw of SNE and 1% of SND groups was significantly increased as compared to the positive control. In conclusion, Spirogyra neglecta (Hassall) Kutzing showed cancer chemopreventive properties at the early stages of diethylnitrosamine-induced hepatocarcinogenesis in rats. Possible inhibitory mechanisms include enhancement of the activities of some detoxifying enzymes and/or suppression of precancerous cells.

Evaluation of hepatic antioxidant capacities of Spirogyra neglecta (Hassall) Kützing in rats.

ABSTRACT Free radicals are one of the causes of chronic and degenerative diseases. Antioxidants can protect the progression of free radical mediated disorders. The aim of this study was to evaluate the antioxidant activity of Spirogyra neglecta (Hassall) Kützing in rats. The rats were divided into 5 groups. Group 1 served as control. Groups 2 and 3 were administered hot water extract of S. neglecta at 50 and 200 mg/kg bw, respectively, while groups 4 and 5 were fed 1% and 4% S. neglecta mixed diet, resp., for 13 weeks. Antioxidant enzymes were evaluated in livers of the rats. The activities of catalase and glutathione reductase were significantly increased in the group fed 50 mg/kg of the extract, compared with the control group. Glutathione peroxidase activity was also significantly higher in the group fed 50 and 200 mg/kg of the extract. The study suggests that S. neglecta may enhance antioxidant systems in the rat liver.

Characterization and immunomodulatory activities of polysaccharides from Spirogyra neglecta (Hassall) Kützing

Sulfated polysaccharides (SP) isolated from freshwater green algae, Spirogyra neglecta (Hassall) Kützing, and fractionated SPs were examined to investigate their molecular characteristics and immunomodulatory activity. The crude and fractionated SPs (F1, F2, and F3) consisted mostly of carbohydrates (68.5–85.3%), uronic acids (3.2–4.9%), and sulfates (2.2–12.2%) with various amounts of proteins (2.6–17.1%). d-galactose (23.5–27.3%), d-glucose (11.5–24.8%), l-fucose (19.0–26.7%), and l-rhamnose (16.4–18.3%) were the major monosaccharide units of these SPs with different levels of l-arabinose (3.0–9.4%), d-xylose (4.6–9.8%), and d-mannose (0.4–2.3%). The SPs contained two sub-fractions with molecular weights (Mw) ranging from 164 × 103 to 1460 × 103 g/mol. The crude and fractionated SPs strongly stimulated murine macrophages, producing considerable amounts of nitric oxide and various cytokines via up-regulation of their mRNA expression by activation of nuclear factor-kappa B and mitogen-activated protein kinases pathways. The main backbone of the most immunoenhancing SP was (1→3)-l-Fucopyranoside, (1→4,6)-d-Glucopyranoside, and (1→4)-d-Galactopyranoside. The SPs stimulated RAW264.7 cells, producing of NO and cytokines via up-regulation of their mRNA expression by activation of NF-κB and MAPKs pathways.