Catalytic function of a tyrosyl residue in tryptophanase

Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane. Pyridoxal 5'-phosphate can prevent this modification efficiently, whereas pyridoxal 5'-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond. The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme. Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the alpha-H of substrates. The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to alpha-H labilization.     

Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp.

A marine photosynthetic bacterium (PS88), identified as Rhodovulum sp., with flocculating ability was isolated from the sea sediment mud of a shrimp cultivation farm in Thailand. This bacterium flocculated in glutamate/malate medium during aerobic dark or anaerobic light cultivation. The flocculating ability was enhanced with the increase of NaCl concentration to 6% (w/v). When PS88 was grown in glutamate/malate medium containing 3.5% NaCl, protein, RNA and DNA were produced exocellularly and there was flocculation. The yields of DNA, RNA and protein were 8.3, 62.5 and 48.5 mg/g dry cell, respectively. The flocculated cells were deflocculated by treatment with a nucleolytic enzyme such as RNase or DNase, while amylase, protease, trypsin, cellulase and pectinase had no deflocculating effect. These results suggest that the exocellular nucleic acids are active in flocculation. Received: 10 April 1998 / Received revision: 14 July 1998 / Accepted: 8 August 1998     

Population ecology of Thrips palmi (Thysanoptera:Thripidae) in orchid farms in Thailand

The population dynamics and in-farm distribution of Thrips palmi Karny were evaluated in two orchid farms, one in the Bangkok metropolitan area and the other in Ratchaburi Province, Thailand. Yellow sticky traps were hung above the orchid canopy (three traps/220 m²), and the trapped adults were counted weekly from November 2009 to October 2010 at the first location and from January to December of 2012 at the second location. T. palmi nymphs and adults were both counted weekly on 300 orchid panicles in an area of 2,200 m² at both locations. We found that the T. palmi population dynamics at the first location was represented by a W-shaped curve, with the distribution clumped throughout the year, whereas the curve was J-shaped at the second location, with the distribution mostly clumped. Qualitative surveys of thrips species were conducted on ten different orchid genera in ten locations and on 20 different Dendrobium hybrids in 20 locations. Five random sampling plots (1 × 4 m²) were examined on each farm. The results indicated that only T. palmi was found on every farm. Moreover, quantitative comparison among five orchid types revealed that Dendrobium flowers with darker color are more attractive to T. palmi. However, this phenomenon does not coincide with Oncidium sp. and Mokara sp. because of their unfavorable morphology for thrips behavior.     

Influence of Oxygen and Glucose on Primary Metabolism and Astaxanthin Production by Phaffia rhodozyma in Batch and Fed-Batch Cultures: Kinetic and Stoichiometric Analysis

The influence of the oxygen and glucose supply on primary metabolism (fermentation, respiration, and anabolism) and astaxanthin production in the yeast Phaffia rhodozyma was investigated. When P. rhodozyma grew under fermentative conditions with limited oxygen or high concentrations of glucose, the astaxanthin production rate decreased remarkably. On the other hand, when the yeast grew under aerobic conditions, the astaxanthin production rate increased with increasing oxygen uptake. A kinetic analysis showed that the respiration rate correlated positively with the astaxanthin production rate, whereas there was a negative correlation with the ethanol production rate. The influence of glucose concentration at a fixed nitrogen concentration with a high level of oxygen was then investigated. The results showed that astaxanthin production was enhanced by an initial high carbon/nitrogen ratio (C/N ratio) present in the medium, but cell growth was inhibited by a high glucose concentration. A stoichiometric analysis suggested that astaxanthin production was enhanced by decreasing the amount of NADPH required for anabolism, which could be achieved by the repression of protein biosynthesis with a high C/N ratio. Based on these results, we performed a two-stage fed-batch culture, in which cell growth was enhanced by a low C/N ratio in the first stage and astaxanthin production was enhanced by a high C/N ratio in the second stage. In this culture system, the highest astaxanthin production, 16.0 mg per liter, was obtained.     

Impedance analysis of bio-fuel cell electrodes

To determine the criteria for the selection of an electrode suitable for a bio-fuel cell (BFC), five electrodes, i.e. silver, aluminum, nickel, stainless steel and carbon fiber cloth were investigated. The performance of the BFC according to the electrode material, including the generated voltage, current density and power density was observed. These results show that the materials used for constructing the electrodes affect the performance of the BFC. An impedance analysis was used to describe the characteristics of the electrodes in the solution. Equivalent circuits of each component such as solution, electrodes-solution interface and electrode were determined from the impedance data. The constant-phase element (CPE) model was applied for data analyzing. It was found that stainless steel, nickel and aluminum behaved like a polarized electrode which has a high electrode-solution interfacial impedance, while carbon fiber cloth and silver had a low impedance like a non-polarized electrode. The impedance data indicated that a higher interfacial impedance will result in a higher loading effect. The results can be summarized that the carbon fiber cloth electrode offers a good electron transfer in the system and thus supplies higher power to the external load.     

Functional role of cysteinyl residues in tryptophanase

Holotryptophanase inactivated by oxidation of cysteinyl residues showed a different absorption spectrum from the native enzyme. At pH 8.0, the native enzyme preferentially existed as a 337-nm species (active form), whereas in the inactive enzyme a 420-nm species (inactive form) was dominant. During the reactivation of the enzyme by reduction with dithiothreitol, an increase at 337 nm and a decrease at 420 nm were observed with concomitant increase in enzymatic activity, which was accompanied by the appearance of two cysteinyl residues per monomer. Specific S-cyanylation of cysteinyl residues by nitrothiocyanobenzoic-acid-inactivated apotryptophanase with the modification of one cysteinyl residue per monomer, whereas holotryptophanase was highly resistant to inactivation with nitrothiocyanobenzoic acid. The essential role of the active-site-bound pyridoxal 5'-phosphate in protection against inactivation was confirmed by the agreement of the K1/2 (protection) of 5.0 microM for pyridoxal 5'-phosphate with Km of 2.0 microM in enzyme catalysis. The inactivation by nitrothiocyanobenzoic acid caused a similar shift in the equilibrium between the 337-nm species and 420-nm species, i.e. decrease of the 337-nm species and increase of the 420-nm species. From the pH dependence of the equilibrium between these two species, pKa of 7.9 and 7.4 was obtained for the inactive and the dithiothreitol-activated enzyme, respectively, indicating that cysteinyl residue(s) participated in lowering the pKa of the interconversion between the 337-nm species (active form) and 420-nm species (inactive form). The possible role of cysteinyl residues in the function of tryptophanase is discussed.     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

Astaxanthin production by Phaffia rhodozymaenhanced in fed-batch culture with glucose and ethanol feeding

Of various carbon sources, examined for the cultivation of Phaffia rhodozyma, ethanol enhanced the astaxanthin content but severely decreased growth. Therefore, high cell mass was obtained by glucose fed-batch culture with pH-stat, and the ethanol feeding was performed based on DO-stat. As a result of this two-stage fed-batch cultivation, 30 g dry cells per liter were obtained, and the astaxanthin content reached 0.72 mg/g, which was 2.2-fold higher than that without ethanol feeding.