Regulation of phenoloxidase activity with sulfur dioxide as the base of production of high-quality instant green tea

The possibility to regulate phenoloxidase activity with sulfur dioxide was studied. It was found that this compound is a potent inhibitor of phenoloxidase of the reversible and mixed type. The inhibitory effect of sulfur dioxide on phenoloxidase provided grounds for a new biotechnological approach to the production of instant green tea. This approach allows increasing the yield of the extractive and the proportion of phenolics in the extractive, thereby improving the organoleptic quality of the product.     

Antibotulinal efficacy of sulfur dioxide in meat.

The addition of sodium metabisulfite as a source of sulfur dioxide delayed botulinal outgrowth in perishable canned comminuted pork when it was temperature abused at 27 degree C. The degree of inhibition was directly related to the level of sulfur dioxide. Levels greater than 100 microgram of sulfur dioxide per g were necessary to achieve significant inhibition when a target level of 100 botulinal spores per g was used. Sodium nitrite partially reduced the efficacy of the sulfur dioxide. Sulfur dioxide offers a new option for the control of botulinal outgrowth in cured or noncured meat and poultry products.     

Sulfur dioxide inhibition of photosynthesis in isolated spinach chloroplasts

Photosynthetic oxygen evolution by isolated spinach (Spinacia oleracea L.) chloroplasts approached complete inhibition in the presence of a 5 mm concentration of sulfur dioxide. A similar inhibition was observed in the presence of equimolar concentrations of bisulfite ions, suggesting a parallel mode of action. In contrast, an equimolar concentration of sulfite ions was markedly less inhibitory and sulfate ions caused negligible inhibition of apparent photosynthesis. The mode of action of sulfur dioxide and related sulfur anions in inhibiting photosynthesis was found to be essentially independent of direct hydrogen-ion effects. Supplements of inorganic pyrophosphate lessened the inhibition of oxygen evolution caused by sulfur dioxide and the sulfur anions.Sulfur dioxide and the sulfur anions were almost equally effective in inhibiting cyclic and noncyclic photophosphorylation in chloroplast suspensions. However, the extent of the inhibition of these photosynthetic reactions does not appear sufficient to account for the inhibition of photosynthetic oxygen evolution by sulfur dioxide.     

Photoreduction of sulfur dioxide by spinach leaves and isolated spinach chloroplasts

Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed.     

10个园林绿化树苗对SO2的反应特性

用开顶式熏气装置对10种2年生园林绿化树苗进行不同浓度的SO2胁迫,并对其叶片气体交换特征参数进行了测定,研究了参试树种对SO2的生理反应及其在绿化中的应用.结果表明:在SO2胁迫条件下,大多数树种的净光合速率(P n)、蒸腾速率(T r)和气孔导度(G s)均出现下降趋势,下降幅度因树种和SO2浓度不同而有较大差异.树种P n与G s、T r与G s之间存在显著线性正相关关系,但是随SO2浓度增加,P n与G s以及T r与G s线性相关的显著程度被削弱,表现出SO2胁迫下不同树种P n变化与G s变化、T r变化与G s变化的复杂性.根据P n下降幅度将树种分为3种类型:轻度敏感(3种)、中度敏感(4种)和高度敏感树种(3种).     

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.Environmental Biology Division     

Effect of Inhibitors on Phenoloxidase of Mycobacterium leprae

Previous results had shown that the human leprosy bacilli possess a phenoloxidase, which, when compared with the enzyme from mammalian and plant sources, seemed unique in the range of substrates utilized and in the nature of the products formed. The effect of several inhibitors on the enzyme in Mycobacterium leprae was tested. Compounds which bind copper were found to be more effective than substrate analogues. Diethyldithiocarbamate penetrated the bacillus and completely suppressed its phenolase activity. Diasone (a derivative of diaminodiphenylsulfone used in the treatment of leprosy) proved to be a potent inhibitor of phenoloxidase of mammalian and plant origin. However, it was less efficient in the case of M. leprae. A biochemical peculiarity of M. leprae was observed in its ability to metabolize mimosine and penicillamine. These compounds produced total inhibition of tyrosinase in melanoma extract and of mushroom tyrosinase. Nontoxic inhibitors of phenoloxidase in the leprosy bacilli may be of value in developing a rational approach to chemotherapy of the disease.     

Isolation of latent phenoloxidase from prepupae of the housefly,Musca domestica

Latent phenoloxidase was purified from prepupae of the housefly, Musca domestica vicina Maquart. The purification procedures included DEAE-cellulose column chromatography, sucrose density gradient centrifugation adn second sucrose density gradient centrifugation. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis in the presence of EDTA. Electrophoresis in the absence of EDTA caused spontaneous activation of latent phenoloxidase. While latent phenoloxidase was fairly stable over the range of temperatures between 0 and 40°C, it was quite sensitive to changes in pH, being stable only around pH 6.0. The molecular weight of latent phenoloxidase was estimated to be 178,000, as determined by gel filtration and sucrose density gradient centrifugation. Furthermore, phenoloxidase formed by the activation of latent phenoloxidase indicated a higher molecular weight (340,000) than that of latent phenoloxidase. Thus, it appears that the mechanism of the activation of latent phenoloxidase involves the association and disassociation system.     

Plant sulfur nutrition: From Sachs to Big Data

Together with water and carbon dioxide plants require 14 essential mineral nutrients to finish their life cycle. The research in plant nutrition can be traced back to Julius Sachs, who was the first to experimentally prove the essentiality of mineral nutrients for plants. Among those elements Sachs showed to be essential is sulfur. Plant sulfur nutrition has been not as extensively studied as the nutrition of nitrogen and phosphate, probably because sulfur was not limiting for agriculture. However, with the reduction of atmospheric sulfur dioxide emissions sulfur deficiency has become common. The research in sulfur nutrition has changed over the years from using yeast and algae as experimental material to adopting Arabidopsis as the plant model as well as from simple biochemical measurements of individual parameters to system biology. Here the evolution of sulfur research from the times of Sachs to the current Big Data is outlined.     

Kinetics and activation energetics of death in Saccharomyces cerevisiae induced by sulfur dioxide

Death of Saccharomyces cerevisiae induced by sulfur dioxide (K(2)S(2)O(2) was used as the SO(2) source) followed saturation kinetics. The enthalpy of activation of death was not affected by concentration over the range tested (5-150) mg/L of (K(2)S(2)O(2) at pH 3.4) and averaged 3.6 x 10(4) cal/mol as compared with 8.5 x 10(4) cal/mol for DeltaH(double dagger) of thermal death. The entropy of activation of death was hyperbolic function of the sulfur dioxide concentration, extrapolated at zero concentration to DeltaS(0) (double dagger) = 36.8 cal mol(-1) K(-1) and tended to DeltaDeltaS(max) (double dagger) = 13.2 cal mol(-1) K(-1) at saturating concentration, yielding a dis0sociation constant of 5.8 x 10(-1) M sulfur dioxide. As was predicted from these results, In K(d) (the specific rate of death induced by sulfur dioxide) was hyperbolic function of concentration under isothermic conditions and extrapolated toa finite value at zero concentration. The Arrhenuis plots and the DeltaS(+/-) plot versus concentration revealed the occurrence of substrate inhabitation of the death effect at high concentrations (above 60 mg/L K(2)S(2)O(2) at pH 3.4). A model is presented involving two types of receptor sites for sulfur dioxide on the cell surface, on directly connected with the death process, the other modulating its entropy of activation.     

NITROGEN FIXATION RATE AND CHLOROPHYLL CONTENT OF THE LICHEN PELTIGERA CANINA EXPOSED TO SULFUR DIOXIDE

In general, the rate of nitrogen fixation decreased when the lichen Peltigera canina (L.) Willd. was exposed to sulfur dioxide gas at levels from 0.1 to 500 ppm; at 30 ppm, however, nitrogen fixation was stimulated. Chlorophyll content decreased as level of sulfur dioxide increased.     

SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.     

Liquid–liquid extraction of fermentation inhibiting compounds in lignocellulose hydrolysate

Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent is suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not suitable for in situ application due to its poor biocompatibility. Biotechnol. Bioeng. 2009;102: 1354–1360. © 2008 Wiley Periodicals, Inc.     

Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans

Protoplasts of Cryptococcus neoformans contain phenoloxidase as a membrane-bound enzyme. The enzyme appeared to be attached on the inner side of cytoplasmic membranes. Synthesis of the enzyme was derepressed by low levels of glucose but was not affected by the level of ammonium. Copper chelators which inhibited the phenoloxidase of other organisms did not affect cryptococcal enzymes. However, cyanide- or iron-chelating agents such as hydroximide derivates or 8-hydroxyquinoline were effective inhibitors, suggesting that cryptococcal phenoloxidase is an iron-containing enzyme. Phenoloxidase of C. neoformans catalyzed the oxidation of various diphenols via dopachrome and labile intermediates to melanin polymers. The kinetic constants (Km) of the phenoloxidase and the permease for dopamine and norepinephrine were low. The correlation between phenoloxidase and the preferential growth of C. neoformans in the host brain is discussed.     

Isolation of saccharolytic dissimilatory sulfate-reducing bacteria

Abstract Four unidentified saccharolytic dissimilatory sulfate-reducing strains were isolated from an anaerobic digester. Cells were Gram-negative, motile, nonsporulating rods which differ markedly from known sulfate reducers especially with respect to carbon source utilisation and sulfur sources which can be reduced. The strains were capable of metabolising at least 26 out of 50 carbohydrates tested. Carbohydrates were, in the absence of exogenous sulfate, fermented to acetate, ethanol, lactate, carbon dioxide and hydrogen. In the presence of excess sulfate carbohydrates were fermented to acetate, ethanol, carbon dioxide, hydrogen and hydrogen sulfide, but lactate was not detected. An oxidized organic or inorganic sulfur source, including elemental sulfur, was not required as a prerequisite for growth on carbohydrates, Lactate was, in the presence of sulfate, converted to acetate, ethanol, carbon dioxide, hydrogen and hydrogen sulfide. In the absence of sulfate no lactate was utilised and no growth was observed.     

The toxic effects of phenoloxidase from the haemolymph of tenebrionid beetles

The injection of haemolymph originating from several species of tenebrionid beetles into blowfly larvae caused a gradual paralysis accompanied by colour changes in the haemolymph of the injected test insects. It was found that the lethal effect of the haemolymph of the beetle Blaps sulcata was due to phenoloxidase. The enzyme was activated by the exposure and incubation of the haemolymph at room temperature.The identity between the toxic factor and phenoloxidase in the beetle's haemolymph was demonstrated by the following data: (1) A correlation between the rate of lethal and phenoloxidase activities during the activation process of the toxic haemolymph. (2) Phenylthiourea, a well-known inhibitor of phenoloxidase, inhibited both the enzymatic and the toxic action of the beetle's haemolymph. (3) A commercial preparation of phenoloxidase (originating from mushrooms) imitated the lethal effects and the accompanying symptoms of the toxic haemolymph. (4) Sephadex G-100 column separation of the Blaps haemolymph revealed a complete overlap between the enzymatic and lethal regions of the elution pattern.The possible effects of phenoloxidase on the haemolymph of the injected insects are discussed.     

Effect of Ethanol,Sulfur Dioxide and Glucose on the Growth of Wine Spoilage Yeasts Using Response Surface Methodology

Response surface methodology (RSM) was used to study the effect of three factors, sulfur dioxide, ethanol and glucose, on the growth of wine spoilage yeast species, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Seventeen central composite rotatable design (CCRD) trials were designed for each test yeast using realistic concentrations of the factors (variables) in premium red wine. Polynomial regression equations were fitted to experimental data points, and the growth inhibitory conditions of these three variables were determined. The overall results showed Sa. ludwigii as the most resistant species growing under high ethanol/free sulfur dioxide concentrations, i.e., 15% (v/v)/20 mg L^-1, 14% (v/v)/32 mg L^-1 and 12.5% (v/v)/40 mg L^-1, whereas other yeasts did not survive under the same levels of ethanol/free sulfur dioxide concentrations. The inhibitory effect of ethanol was primarily observed during longer incubation periods, compared with sulfur dioxide, which showed an immediate effect. In some CCRD trials, Sa. ludwigii and S. cerevisiae showed growth recovery after a short death period under the exposure of 20–32 mg L^-1 sulfur dioxide in the presence of 11% (v/v) or more ethanol. However, Sc. pombe and Z. bailii did not show such growth recovery under similar conditions. Up to 10 g L^-1 of glucose did not prevent cell death under the sulfur dioxide or ethanol stress. This observation demonstrates that the sugar levels commonly used in wine to sweeten the mouthfeel do not increase wine susceptibility to spoilage yeasts, contrary to the anecdotal evidence.     

Sulfur-dependent inhibition of protein and RNA synthesis by iron-grown Thiobacillus ferrooxidans

The addition of sulfur to iron-grown Thiobacillus ferrooxidans resulted in a rapid inhibition in the rates of protein synthesis and RNA synthesis. The inhibition of both functions was measured within 15 to 30 min and was maximal between 70 and 90% compared to the iron-grown controls. DNA synthesis, carbon dioxide fixation, and short-term ferrous oxidation rates of the bacteria growing on ferrous ions were not effected by sulfur addition, indicating that the sulfur addition was not perturbing general cellular energy metabolism. The inhibition caused by sulfur mimicked the effect of the RNA synthesis inhibitor, rifampicin, which inhibited both RNA and protein synthesis, but did not correspond with the translational inhibitor, chloramphenicol, which inhibited only protein synthesis in the first hour. Since chloramphenicol pretreatment did not block the sulfur effect, the inhibition of RNA synthesis following sulfur addition was not mediated through protein synthesis.     

Insect melanogenesis. III. Metabolon formation in the melanogenic pathway-regulation of phenoloxidase activityy by endogenous dopachrome isomerase (decarboxylating) from Manduca sexta

Tyrosinase initiates melanogenesis in a variety of organisms. The nature of melanin formed is modified subsequently by dopachrome isomerase and other melanogenic proteins. Earlier, we reported the partial purification of dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta and demonstrated the generation of a new quinone methide intermediate during melanogenesis (Sugumaran, M., and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). In this paper, we report the purification of this enzyme to homogeneity and a novel inhibition mechanism for regulation of phenoloxidase activity. The activity of phenoloxidase isolated from M. sexta was markedly inhibited by purified dopachrome isomerase. In turn, phenoloxidase also reciprocated by inhibiting the isomerase activity. Preformed dopaminechrome did not serve as the substrate for the isomerase; but dopaminechrome that generated in situ by phenoloxidase was readily converted to melanin pigment by the phenoloxidase/isomerase mixture. Furthermore, the isomerase, which has a molecular weight of about 40,000 in native state, exhibited retardation during affinity electrophoresis on sodium dodeyl sulfate (SDS)-polyacrylamide gel electrophoresis gel copolymerized with tyrosinase and migrated with a molecular weight of 50,000, indicating complex formation with phenoloxidase. Electrophoresis of pupal cuticular extract on polyacrylamide gel, followed by activity staining revealed the presence of a protein band carrying both phenoloxidase and isomerase activity. Accordingly, a high-molecular-weight melanogenic complex was isolated from the pharate cuticle of M. sexta. The complex catalyzed the generation of melanochrome from dopa, while the free phenoloxidase produced only dopachrome from the same substrate. When the complex was treated with trace amounts of SDS, which inhibited the activity of dopachrome isomerase present in the complex, then only the conversion of dopa to dopachrome was observed. These studies confirm the formation of a melanogenic complex between phenoloxidase and dopachrome isomerase. By forming a complex and regulating each other's activity, these two enzymes seem to control the levels of endogenous quinones.     

The effects of sulfur dioxide and A2 influenza virus on pneumonia and weight reduction in mice: an analysis of stimulus-response relationships

Using a mouse-influenza model, mice were exposed in exposure chambers to various doses of influenza A₂ virus and sulfur dioxide, each alone and in combination. Temperature, relative humidity and length of exposure were all controlled. Control animals were in the same setting, but did not have influenza virus or sulfur dioxide exposure. The two responses measured were: the amount of pneumonia produced and the amount of weight reduction produced by the exposure stimuli. A stimulus-response method of analysis was utilized.Virus exposure produced pneumonia even at low dose. Sulfur dioxide produced pneumonia at higher doses, but appeared to have an inverse effect at lower doses when compared to control animals. The two combined produced more pneumonia than either one did separately. Sulfur dioxide produced more weight reduction than influenza virus, and combination exposures produced more weight reduction than either stimulus did separately. Dosage effects (correlations) were seen in both the production of pneumonia and the production of weight reduction. The stimulus-response method clarified and quantified the relationships found.