Presence and physiologic regulation of alcohol oxidase activity in an indigenous fungus isolated from petroleum-contaminated soils

A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde. Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2). Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20, 38, and 46 kDa that could be part of the native enzyme. In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha. The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol. K _m values obtained for methanol and hexa-decanol indicated a higher affinity for the latter. Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.     

Effects of carbon source on expression of alcohol oxidase activity and on morphologic pattern of YR-1 strain, a filamentous fungus isolated from petroleum-contaminated soils

Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homogenization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose. In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2. In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE. New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively. The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons. The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

A different method of measuring and detecting mono-and dioxygenase activities

A spectrophotometric method of measuring oxygenase activity in cell extracts or in zymograms was developed. It is an easy and cheap method that allows spectrophotometric measurement of activity by a colored reaction and reveals activity bands in a polyacrylamide gel electrophoresis (PAGE) gel as brown bands. To prove its usefulness, we report on a study with the oxygenase present in strain YR-1, isolated from petroleum-contaminated soils, that uses hydrocarbons as its sole carbon source. Soluble oxygenase activity was detected (under our conditions of cellular homogenization) in the mycelium of a filamentous fungus strain named YR-1. Oxygenase activity from aerobically grown mycelium was detected in growth medium containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited similar optimum pH for the hydroxylation of different aliphatic or aromatic substrates (decane, hexadecane, benzene, and naphthalene) to the corresponding alcohols. Zymogram analysis conducted with partially purified fractions from cell extracts from the aerobic mycelium of the YR-1 strain indicated the existence of only one oxygenase enzyme. Partially purified samples of enzyme, analyzed by sodium dodecyl sulfate PAGE, indicated the presence of one major protein band with a mol wt of 56 kDa that can be a constituent of the native enzyme. In samples of the enzyme, the 56-kDa protein gave a positive reaction in immuno-detection experiments with antibodies directed against oxygenase from soybean. The partially purified enzyme oxidized different substrates, although higher activity was displayed with benzene. K _m values obtained for benzene and decane indicated a higher affinity for the latter     

Analysis of the bioactivity of magnetically immunoisolated peroxisomes

Peroxisomes produce reactive oxygen species which may participate in biotransformations of innate biomolecules and xenobiotics. Isolating functional peroxisomes with low levels of contaminants would be a useful tool to investigate biotransformations occurring in these organelles that are usually confounded with biotransformations occurring in other co-isolated organelles. Here, we immunoisolate peroxisomes and demonstrate that the impurity level after isolation is low and that peroxisomes retain their biological activity. In this method, an antibody targeting a 70-kDa peroxisomal membrane protein was immobilized to silanized magnetic iron oxide beads (1–4 μm in diameter) coated with Protein A. Peroxisomes from L6 rat myoblast homogenates were magnetically captured, washed, and then analyzed for subcellular composition using enzymatic assays. Based on the ratio of peroxisomal to lysosomal activity, the retained fraction is 70-fold enriched relative to the unretained fraction. Similarly, the ratio of peroxisomal activity to mitochondrial content suggests that the retained fraction is >30-fold enriched relative to the unretained fraction. H₂O₂ production from the β-oxidation of palmitoyl-CoA demonstrated that the isolated peroxisomal fraction was biologically active. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis confirmed that the immunopurified fractions were capable of transforming the anticancer drug doxorubicin and the fatty acid analog, BODIPY 500/510 C1C12. Besides its use to investigate peroxisome biotransformations in health and disease, the combination of magnetic immunoisolation with CE-LIF could be widely applicable to investigate subcellular-specific biotransformations of xenobiotics occurring at immunoisolated subcellular compartments.     

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.     

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation.

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using 125I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5'-nucleotidase, Mg2+-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-gamma-32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg2+-ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+-stimulated phosphatase activity does not correlate with Mg2+-ATPase, the two assay systems define separate enzymatic processes.     

Subzelluläre Verteilung von Enzymen des Purinabbaues in pflanzlichen Speicherorganen

Zusammenfassung Die Verteilung von Uratoxidase (E.C. 1.7.3.3), Allantoinase (E.C. 3.5.2.5) und Allantoicase (E.C. 3.5.3.4) in subzellulären Fraktionen aus Kartoffelknollen und Kotyledonen von keimenden Kürbis- und Erbsensamen wurde untersucht. Uratoxidase und Allantoinase wurden in allen drei Pflanzen nachgewiesen, Allantoicase nur in Kartoffeln. Die Isolierung und Charakterisierung von Glyoxysomen aus Kürbiskotyledonen und von Peroxisomen aus Erbsenkotyledonen wird beschrieben. In allen drei Zelltypen war ein größerer Teil der Uratoxidaseaktivität in den Peroxisomen vorhanden. Diese enthielten auch einen signifikanten Teil der Allantoinaseaktivität in Erbsen- und Kürbissamen. Der Rest der Enzymaktivitäten wurde im 10 000g-Überstand der differentiellen Zentrifugation nachgewiesen.

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Subcellular distribution of enzymes of purin degradation in storage organs from plants

The distribution of urate oxidase (E.C. 1.7.3.3), allantoinase (E.C. 3.5.2.5), and allantoicase (E.C. 3.5.3.4) was investigated in subcellular fractions from potato tubers and from cotyledons of germinating seeds of pumpkins and peas. Urate oxidase and allantoinase were detected in all three plants, allantoicase only in potatoes. The isolation and characterization of glyoxysomes from pumpkin cotyledons and of peroxisomes from pea cotyledons is described. In all three tissues a considerable part of the urate oxidase activity was present in the peroxisomes, which contained also a significant part of the allantoinase activity of pea and pumpkin seeds. The rest of the enzyme activities was detected in the 10 000g supernatant of the differential centrifugation.

     

Environmental biodegradation of synthetic polymers I. Test methodologies and procedures

Biodegradation of synthetic polymers is an important property that is used in many applications. Evaluation of the extent of biodegradation has used different methods in recent years. For each environmental compartment, different approaches have to be made in order to obtain valuable data on biodegradability.This review describes validated and accepted methods based on standardized biodegradation tests, analytical tests, enzymatic tests or tests of physical properties to evaluate the biodegradability of synthetic polymers for different types of environmental compartments (e.g., soil, compost or aqueous media).Part II of this review will subsequently report on the environmental biodegradation of different groups of synthetic polymers.     

Genome Annotation and Validation of Keratin-Hydrolyzing Proteolytic Enzymes from <Emphasis Type="Italic">Serratia marcescens</Emphasis> EGD-HP20

Stabilization and utilization of poultry waste demand efficient biodegradation either by mixture of enzymes or by microbial system that can produce different types of protein-hydrolyzing enzymes. For utilization of this keratinous biomass, in the present study, genome was sequenced and annotated for a bacterium having multiple enzymatic options for hydrolysis of different soluble and insoluble protein fractions of poultry waste. Among the soluble protein substrates, optimum production of enzyme and soluble protein was observed in case of casein, whereas among the insoluble protein substrates, maximum production of enzyme was achieved when broken nails were used. Conditions for enhanced enzyme activity with concurrent degradation of keratin-rich poultry feather waste to protein-rich hydrolysate were optimized for different growth parameters. The bacterium grew well and highest protease production occurred in 144 h at mesophilic temperature (30 °C) and alkaline condition (pH 8–10) with enzyme activities of 134 and 168 U/mL, respectively.     

Alcohol sensing membrane based on immobilized ruthenium(II) complex in carboxylated PVC and surface covalently bonded alcohol oxidase

Alcohol sensing membranes coated on overhead transparency films for the continuous monitoring of ethanol, propanol and butanol are presented. Alcohol oxidation catalyzed by alcohol oxidase in conjunction with the fluorescence quenching reaction of oxygen-sensitive dye ion-pair, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) didodecylsulphate was chosen for the determination. Alcohol oxidase was immobilized covalently on a plasticized carboxylated poly(vinyl chloride) membrane and the oxygen-sensitive dye ion-pair was entrapped in the same membrane. The sensing membrane relates oxygen consumption, as a result of enzymatic oxidation, to alcohol concentration. Measurements have been performed in air-saturated alcohol standard solutions of pH 7.0. Storage stability, reproducibility and the effect of pH on sensing membrane performance have been studied in detail. The alcohol sensing membrane proposed here is simple to prepare and has a fairly rapid response time of <1 min. It has been successfully applied to the determination of the ethanol contents in various spirits.     

Effects of ultraviolet light irradiation in biotreatment of organophosphates

Excess biomass accumulation in reactor biodegradation processes is undesirable: it increases the disposal cost and upsets the operation of biological reactors if not properly controlled. In this study, we investigated the feasibility of using ultraviolet (UV) light irradiation to reduce biomass accumulation and increase the specific biodegradation activity. UV irradiation has been widely used to introduce DNA damage in bacteria. Here we apply this technology to the biodegradation of organophosphates by recombinantEscherichia coli strains that contain arecA mutation and a cloned organophosphate hydrolase gene. We show that therecA negative strains after UV irradiation reduce the growth rate but increase the specific organophosphate hydrolase activity. This increase in specific enzyme activity is not owing to continued protein synthesis from the plasmid after the damage of chromosomal DNA by UV irradiation. Rather, it is likely to be caused by an increase in membrane permeability to the substrate. Kinetic analysis suggests that the membrane transport of paraoxon is the rate-limiting step in its biodegradation.     

Enzymatic Biosensors Based on Carbon Nanotubes Paste Electrodes

The performance of enzymatic biosensors based on the immobilization of different enzymes within a carbon nanotubes paste electrode (CNTPE) prepared by dispersion of multi‐wall carbon nanotubes (MWNT) and mineral oil is reported in this work. The strong electrocatalytic activity of carbon nanotubes towards the reduction of hydrogen peroxide and quinones and the oxidation of NADH have allowed an effective low‐potential amperometric determination of lactate, phenols, catechols and ethanol, in connection with the incorporation of lactate oxidase, polyphenol oxidase and alcohol dehydrogenase/NAD⁺, respectively, within the composite matrix. Compared to the analogous enzymatic CPEs, a great enhancement in the response is observed at the enzymatic CNTPEs. Therefore, highly sensitive lactate, phenols, catechols and alcohols biosensors without using any metal or redox mediator can be obtained with this new composite material.     

An electron microscopic and enzymic study of rat liver peroxisomal nucleoid core and its association with urate oxidase

The appearance of the characteristic crystalloid core of rat liver peroxisomes is emulated by the electron microscopic (EM) appearance of highly purified urate oxidase prepared from the same tissue. The purity of the enzyme preparation was established by gel electrophoresis under various conditions and the specific enzyme activity was at least as high as any previously reported. The amino acid composition of urate oxidase was determined. As additional evidence for close association of the peroxisomal core with urate oxidase, it was demonstrated that the biphasic changes in rat liver urate oxidase activity in response to prolonged starvation were paralleled by changes in the EM appearance of peroxisomes. Under comparable conditions catalase, another peroxisomal enzyme, did not show the same changes in activity as did urate oxidase. Evidence for the possible identity of urate oxidase with the peroxisomal crystalloid of rat liver has been presented, all materials having been obtained from, and experiments performed with, the rat.     

小分子玉米肽Leu-Asp-Tyr-Glu保护线粒体抗氧化损伤的研究

线粒体是细胞内氧代谢的主要场所,其膜上富含不饱和脂肪酸,使得线粒体成为自由基进攻的主要靶部位^[1].自由基作用于线粒体膜发生脂质过氧化损伤反应,引起膜的结构与功能改变,使得膜上一系列重要的酶空间排列紊乱,活性降低,膜的通透性改变,诱发多种疾病^[2].目前,抗氧化肽类作为一种非酶类自由基清除剂的研究正在兴起^[3].来源于玉米醇溶蛋白酶降解物中的低分子量肽具有许多重要的生理功能,如促进乙醇代谢、抗疲劳作用以及降血脂等功效^[4].而这些功能的实现主要依赖于其抗氧化活性^[5].最近,我们从玉米蛋白酶解物中获得了一种新的小分子抗氧化肽Leu-Asp-Tyr-Glu(LD YE,LD YE经文献检索为一种新的寡肽)^[6].     

BINDING OF GLYCOLATE OXIDASE TO PEROXISOMAL MEMBRANE AS AFFECTED BY LIGHT

The stability of the bond of glycolate oxidase to the peroxisomal membrane obtained from etiolated barley leaves, (Hordeum vulgare L. var Dvir) is affected by the pH of the homogenization medium. Peroxisomes isolated at pH 4 or 10 retain glycolic oxidase, while peroxisomes isolated at pH 69 loose this enzyme during centrifugation. Preillumination of whole seedlings, detached leaves or even homogenates changes the effect of pH on the retention of glycolate oxidase-attachment which is strengthened at pH 9–10 and becomes very loose at pH 4–7. White and red light are responsible for the effect described. Far red light reverses this effect of red light. Illumination induces a change in the isoelectric point (IpH) of peroxisomes without affecting the IpH of soluble enzymes. This red light-induced change on IpH of microbodies is not reversed by far red light. The possible effects of light on peroxisomal membrane properties and on modulation of glycolate oxidase binding are discussed.     

Study on stereospecificity of enzyme reaction related to peroxisomal bile acid synthesis in rat liver.

We examined stereoselectivities of enzymes related to bile acid formation in hepatic peroxisomes using two stereoisomers of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and its coenzyme A (CoA) derivatives. The activity of acyl-CoA synthetase for 25 S-THCA was 1.4-times higher than that for 25 R-THCA. The difference was also observed after clofibrate-treatment. This activity was located in microsomes, differing from palmitoyl-CoA synthetase located in mitochondria, peroxisomes and microsomes. There was no stereoselectivity in the reaction of peroxisomal fatty acyl-CoA oxidase for THCA isomers, and the activity was one tenth of that for acyl-CoA synthetase. Considering the overall reaction of peroxisomal bile acid formation, the stereoselective difference observed in THCA-CoA synthesis should be denied. Thus, the previous finding that the overall formation of bile acid from THCA was not stereoselective was further confirmed. Furthermore, the activity for THCA oxidation was not induced by clofibric acid, suggesting that there would be different isozymes of peroxisomal acyl-CoA oxidase against THCA-CoA and palmitoyl-CoA.     

Yeast cells sucrose biosensor based on a potentiometric oxygen electrode

A new microbial biosensor based on an immobilised microorganisms (Saccharomyces cerevisiae) and a potentiometric oxygen electrode is described. Determination is based on the respiratory activity of the microorganism in presence of different sugars (sucrose and glucose). A response time of ca. 4 min for the steady-state method and 2 min for the initial slope method was obtained. Potentiometric detection has the advantage of an extended calibration range and a low detection limit. The calibration curve for sucrose was linear in the range 1×10⁻⁵ to 3×10⁻² M. This biosensor was used for selective monitoring of sucrose in the presence of glucose, using a second anti-interference enzymatic layer with glucose oxidase (GOD) and catalase (CAT). Interference of glucose in the determination of sucrose decreases from 15% for a microbial biosensor to a maximum 3.5% for the hybrid biosensor. The hybrid biosensor was used to determine sucrose in soft drinks. A good correlation between the results for the biosensor and a spectrophotometric method with dinitrosalicylic acid was achieved.     

Catalysis of the electrochemical oxidation of glucose by glucose oxidase and a single electron cosubstrate: kinetics in viscous solutions

Catalysis of the electrochemical oxidation of glucose by glucose oxidase with a single electron mediator (cosubstrate) may be used to transform mixtures of concentrated industrial sugars. How the high viscosity of such media may affect the enzymatic reaction and the transport of the mediator can be mimicked by addition of large concentrations of sucrose to glucose solutions. Cyclic voltammetry then provides a simple means of investigating the effect of an increased viscosity on the kinetics of the enzymatic reaction and the diffusion of the mediator. The diffusion coefficient of the mediator is decreased 10 times by addition of 1.6 M sucrose. At pH 8, in the presence of the same concentration of sucrose, the catalytic activity of the enzyme towards its substrate is only slightly affected. A 35% decrease of the glucose Michaelis constant is observed. The reaction of the reduced enzyme with the cosubstrate is six times slower and the mediator Michaelis constant undergoes a three-fold increase. It follows that glucose oxidase remains an efficient catalyst in such viscous media.     

Decolorization and Biodegradation of Rubine GFL by Microbial Consortium GG-BL in Sequential Aerobic/Microaerophilic Process

This study represents the development of a new batch method by consortium GG-BL using two microbial cultures viz., Galactomyces geotrichum MTCC 1360 and Brevibacillus laterosporus MTCC 2298, by varying environmental conditions for the decolorization and biodegradation of Rubine GFL. Consortium was found to give better decolorization and degradation of Rubine GFL as compared to the individual microorganism at aerobic/microaerophilic process. The consortial metabolic activity of these strains lead to 100% decolorization of Rubine GFL (50?mg/L) within 30?h with significant reduction in chemical oxygen demand (79%) and total organic carbon (68%). Induction in the activities of laccase, veratryl alcohol oxidase, tyrosinase, azo reductase, and riboflavin reductase suggested their role in the decolorization process. Nondenaturing polyacrylamide gel electrophoresis analysis showed differential induction pattern of oxidoreductive enzymes during decolorization of the dye at different incubation temperatures. The degradation of Rubine GFL into different metabolites by individual organism and in consortium was confirmed using high performance thin layer chromatography, high performance liquid chromatography, Fourier transform infrared spectroscopy, and gas chromatography-mass spectroscopy analysis. Phytotoxicity studies revealed nontoxic nature of the metabolites of Rubine GFL.