Biodegradation of p-aminoazobenzene by Bacillus subtilis under aerobic conditions

Biological oxidation of organic dyes is important for textile industry wastewater treatment. The aim of this work was to assess the biodegradation kinetics of a specific azo-dye, p-aminoazobenzene. The degradation of p-aminoazobenzene by Bacillus subtilis was examined through batch experiments in order to investigate the effect of p-aminoazobenzene on the bacterial growth rate and elucidate the mechanism of dye degradation. The results proved that B. subtilis cometabolizes p-aminoazobenzene in the presence of glucose as carbon source, producing aniline and p-phenylenediamine as the nitrogen–nitrogen double bond is broken. The azo-dye was found to act as an inhibitor to microbial growth. A mathematical model was developed that describes cellular growth, glucose utilization, p-aminoazobenzene degradation and product formation. Received 26 July 1996/ Accepted in revised form 14 May 1997     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

SUBCELLULAR LOCALIZATION OF TRYPTOPHAN-5-MONO-OXYGENASE IN BOVINE PINEAL GLANDS AND RAPHE NUCLEI

Abstract— Tryptophan-5-mono-oxygenase from both bovine raphe nuclei and pineal glands was activated by preincubation with dithiothreitol and ferrous ion at pH 8.5. The optimum pH for the enzyme activity lies between pH 6.4 and 7.3. Preincubation increased the activity of the enzyme from raphe nuclei by about 20 times in both the homogenate and 105,000 g precipitate prepared from it. Activity in the 105,000 g supernatant fraction was about trebled. Corresponding increases in pineal gland enzyme activity were noted: 100 times in homogenate and 105,000 g precipitate and 15 times in 105,000 g supernatant fluid. Total recoveries of activated enzyme from the homogenate prepared in hypo-osmotic medium, in the 105,000 g supernatant and precipitate, were 87.1% and 79.0% for raphe nuclei and pineal glands respectively. Of this, 89.5-91.3% in the case of the raphe nuclei and 76.0-82.0% in the case of the pineal glands, was found in the precipitate. In contrast, 85-90% of the lactate dehydrogenase activity was found in the supernatant fraction. The results of subcellular fractionation revealed that the raphe nuclear enzyme was located in both 'mitochondrial' and 'microsomal' fraction while the pineal gland enzyme was effectively restricted to the 'mitochondrial' fraction. The structural characteristics of the fraction were confirmed by electron microscopy.     

Acid phosphatase activity in maize leaves as related to their evolution and phosphorus deficiency

The effect of phosphorus deficiency on the activity of acid phosphatase of the first, second and third leaves of maize plants was followed. The supernatant obtained by centrifuging the homogenate of plant tissue at 1500 ×g was further centrifuged at 18 000 ×g, the sediment marked as fraction II and the supernatant as fraction III. Acid phosphatase activity of fraction II of the first to third leaves was for the whole period of culture higher in plants grown in the nutrient solution without phosphate. In fraction III this relation was established in the first leaf, after 3 days of culture in the second leaf and after 5 days in the third leaf. In all leaves higher enzyme activity was unambiguously determined in fraction III when compared with fraction II. Higher acid phosphatase activity was established in those leaves which were younger in their development, particularly in the first days of culture. With the ageing of leaves the enzyme activity decreased.     

Properties of an N,N-dimethyl-p-aminoazobenzene Oxide Reductase Purified from Rat Liver Cytosol

Homogenates of all rat tissues examined, except brain, catalyze reduction of N,N-dimethyl-p-aminoazobenzene N-oxide (DMAB N-oxide) to N,N-dimethyl-p-aminoazobenzene by NADPH. Liver is the most active, and about one third of the homogenate activity of this tissue is recovered in the cytosol fraction. The purified cytosol enzyme has the properties of a tetrameric protein (Mr 370,000) consisting of identical subunits free from chromophores that absorb in the visible spectrum and from metals or other detectable prosthetic groups. The purified reductase is also free from NADPH oxidase and from cytochrome c or azo reductase activities. The enzyme is quite specific for NADPH as reductant and DMAB N-oxide as the electron acceptor. Reduction of other N,N-dimethyl-arylamine or alkylamine oxides as well as N-methylheterocyclicamine oxides could not be detected. Analysis of kinetic data indicate that, at saturating concentrations of the other substrate, 21 μM NADPH and 700 μM DMAB N-oxide are required for half maximal velocity. At infinite concentrations of both substrates the turnover is 150 min^?1 at 37 °C.     

How Can a Yeast Catalyze Enantioselective Protonation?

In the enantioselective hydrolysis of enol esters with Pichia farinosa IAM 4682 to give α-chiral ketones, the final enantioselective protonation was found to be promoted by a factor differed from the enzyme catalyzing simple hydrolysis. The crude cell-free extracts from P. farinosa was subjected to ultracentrifugation. Although the supernatant fraction could hydrolyze 1-acetoxy-2-benzylcyclohexene (1), the resulting 2-benzylcyclohexanone (2) was a racemate. On the other hand, the precipitate could not hydrolyze 1. However, on mixing of both fractions the suspension recovered again an enantioselective ability effectively to afford optically active (R)-2. The same phenomena were observed in the hydrolysis using commercially available lipases and an esterase. These results indicate that enantioselectivity-promoting factor should be involved in the precipitate.     

Localization of acid phosphatase activity in maize root under phosphorus deficiency

The effect of phosphorus deficiency on acid phosphatase activity in the apical, middle and basal parts of the root of maize plants was followed. The supernatant obtained by centrifuging the homogenate of plant tissue at 1500 ×g was further centrifuged at 18 000 ×g, the sediment being marked as fraction II and the supernatant as fraction III. The results obtained document the fact that acid phosphatase activity of the two fractions of all analyzed root segments was higher in plants cultured in nutrient medium without phosphate than in those containing phosphorus in nutrient medium. In most cases this difference was significant to highly significant. The results of experiments proved unambiguously a higher enzymatic activity in all root segments in fraction III than in fraction II. In fraction III the highest acid phosphatase activity was found in the apical part, in fraction II in the basal part of the root.     

The nature of the differential inhibition of polyphenylalanine synthesis in extracts from Artemia salina and rabbit reticulocytes by the Phytolacca americana protein

The difference in sensitivity of polyphenylalanine synthesis in extracts from Artemia salina and rabbit reticulocytes to inhibition by the Phytolacca americana protein (PAP) has been found to be linked to the source of the supernatant enzyme fraction and not the ribosomes. In the presence of reticulocyte supernatant enzyme fraction polyphenylalanine synthesis is less sensitive to inhibition by PAP than that observed in the presence of A. salina supernatant enzyme fraction. The results suggest that reticulocyte elongation factor 2 is responsible for this effect.     

Gene Alterations in Paired Supernatants and Precipitates from Malignant Pleural Effusions of Non-Squamous Non-Small Cell Lung Cancer

OBJECTIVE: This study investigated the feasibility of using malignant pleural effusion (MPE) supernatant and paired cell blocks (precipitate) for gene profiling in patients with non-small cell lung cancer (NSCLC) using next-generation sequencing (NGS) technique. METHODS: Stage IV non-squamous NSCLC patients with MPE were eligible in this prospective study and recruited from Zhejiang Cancer Hospital between May 2014 and October 2015. MPE supernatant and paired precipitate sample gene alterations were determined with NGS containing 14 cancer-related genes. Progression free survival (PFS) was evaluated using Kaplan–Meier method and compared using log-rank test. RESULTS: A total of 102 patients were enrolled in the present study. All pleural effusions were confirmed as malignant with cytological smears. A total of 77 paired MPE supernatant and precipitate samples were acquired from the 102 patients. The results revealed that there were no statistically significant differences in the detection rate and maximum allelic fraction between supernatant and precipitate samples (P = 1.0 and P = .6). Collectively, 172 and 158 genomic alterations with 112 shared mutations were identified in supernatant and precipitate samples, respectively. Comparable PFS was found in EGFR mutation patients according to the supernatant and precipitate sample results (14.0 vs.13.9 months, P = .90). CONCLUSIONS: These results demonstrated that MPE supernatants were comparable to precipitate samples for detection of genetic alterations. However, gene mutation heterogeneity was found between these two media types.     

Peroxidase activity in mycelia of Aspergillus parasiticus that degrade aflatoxin

Summary Extracts of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were assayed for peroxidase activity and for their ability to degrade aflatoxin. A positive relationship existed between rates of aflatoxin degradation and amount of peroxidase activity in these extracts. The supernatant fluid of homogenates from mycelia grown under similar conditions varied in amount of peroxidase present (170 to 2215 U/g). The fraction obtained, by precipitation with (NH₄)₂SO₄ at 45% of saturation, from six different homogenates prepared from three mycelial mats contained peroxidase and degraded aflatoxin. Rates of aflatoxin degradation by and amounts of peroxidase activity in each sample obtained from mycelial homogenates with (NH₄)₂SO₄ at 60% of saturation varied; however, when increased amounts of peroxidase activity were present, more aflatoxin was degraded and vice versa. Relatively little peroxidase activity was present in the fraction obtained with (NH₄)₂SO₄ at 30% of saturation and little or no aflatoxin was degraded by this precipitate. Trends for degradation of aflatoxin when more or less peroxidase activity was present in mycelial preparations suggest that the enzyme may be involved in degradation of aflatoxin by the Aspergillus.     

Aminopeptidases in the Acidic Fraction of the Yeast Autolysate

Two aminopeptidases, I and II, were found in the acidic fraction of the yeast autolysate, adsorbed on DEAE-cellose and DEAE-Sephadex A&50. Aminopeptidase I was purified as a single protein with a molecular weight of 200,000. The enzyme required Zn for its activity and hydrolyzed dipeptides, and a polypeptide (glucagon). It also hydrolyzed amides, naphthylamides and the p-nitroanilide of amino acids. The enzyme was strongly inhibited by sulfhydryl reagents. Aminopeptidase II seemed also to be a metal enzyme with a molecular weight of 34,000. The enzyme hydrolyzed the dipeptide and tetrapeptide but not leucine-p-nitroanilide.     

Defects in a proteolytic step of light-harvesting complex II in an<Emphasis Type="Italic">Arabidopsis</Emphasis> stay-green mutant,<Emphasis Type="Italic">ore10</Emphasis>, during dark-induced leaf senescence

During dark-induced leaf senescence (DIS), the non-functional stay-green mutantore10 showed delayed chlorophyll (Chl) degradation and increased stability in its light-harvesting complex II (LHCII). These phenomena were closely related to the formation of aggregates that mainly consisted of terminal-truncated LHCII (Oh et al., 2003). Theore10 mutant apparently lacks the protease needed to degrade the truncated LHCII. In wild-type (WT) plants, protease was found in the thylakoid fraction, but not the soluble fraction. A similar experiment using dansylated LHCII revealed that the protease degraded both WT andore10 LHCII, indicating that its stability inore10 perhaps did not result from a defect in the LHCII polypeptides themselves. Although protease activity was not present in non-senesced WT leaves, it was induced during DIS. It also was possible to diminish the high level of protease present in the thylakoids through high-salt washing, suggesting that this enzyme is extrinsically bound to the outer surface of the stroma-exposed thylakoid regions.     

Subcellular and regional localization ofγ-glutamyl transpeptidase in sheep brain

Studies of the subcellular distribution of-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity when-glutamylp-nitroanalide was used as substrate. This activity, however, represented only 5% of the total-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments.The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a highK _mand the other a lowK _m for the substrate,-glutamylp-nitroanilide.It was observed that the enzyme-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 molp-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

Dominance of iminopeptidase activity in the human oral bacteriumTreponema denticola ATCC 35405

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 M p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.     

Degradative pathway of p-aminoazobenzene by Bacillus subtilis

Summary p-Aminoazobenzene was degraded by Bacillus subtilis to aniline and p-phenylenediamine by reductive fission of an azo bond. The aniline was then acetylated to acetanilide while the p-phenylenediamine underwent 2 successive acetylations to yield p-aminoacetanilide and p-phenylenediacetanilide. In addition, another pathway was found in Bacillus subtilis in which p-aminoazobenzene was metabolised to p-acetamidoazobenzene.     

A fibre cocktail of fenugreek, guar gum and wheat bran reduces oxidative modification of LDL induced by an atherogenic diet in rats

Background LDL (low-density lipoprotein) oxidation is a key trigger factor for the development of atherosclerosis. Relatively few studies exist on the impact of dietary fibre on LDL oxidation. This study was undertaken to evaluate the influence of a novel fibre mix of fenugreek seed powder, guar gum and wheat bran (Fibernat) on LDL oxidation induced by an atherogenic diet. Method Male Wistar albino rats were administered one of the following diets: (1) a control diet that was fibre-free (Group I); (2) an atherogenic diet containing 1.5% cholesterol and 0.1% cholic acid (Group II) or (3) an atherogenic diet supplemented with Fibernat (Group III). Peroxidative changes in low-density lipoprotein (LDL) and the oxidative susceptibility of LDL and the LDL + VLDL (very low-density lipoprotein) fraction were determined. As a corollary to the oxidative modification theory, the titer of autoantibodies to oxidised LDL (oxLDL) was determined at various time points of the study. In addition, plasma homocysteine (tHcy) and lipoprotein (Lp (a)), apolipoprotein (apoB), cholesterol, triglyceride, phospholipid and α-tocopherol content of LDL were determined. Results A decrease in malonaldehyde (MDA) content (p < 0.05) and relative electrophoretic mobility (REM) of LDL was observed in the group III rats as compared to the group II rats. An increase in lag time to oxidation (p < 0.01) and decrease in maximum oxidation (p < 0.01) and oxidation rate (p < 0.01) were observed in the LDL + VLDL fraction of group III rats. In group II rats, formation of autoantibodies to oxLDL occurred at an earlier time point and at levels greater than in the group III rats. Fibernat, had a sparing effect on LDL α-tocopherol, which was about 51% higher in the group III rats than in the group II rats; apo B content of LDL was reduced by 37.6% in group III rats. LDL of group III rats displayed a decrease in free and ester cholesterol (p < 0.01) as compared to that of group II. A decrease in plasma homocysteine (p < 0.01) and an increase in GSH (p < 0.05) were also observed in group III rats when compared with that of group II. Conclusion Fibernat administration appears to combat oxidative stress resulting in a trend to lower oxidative modification of LDL. In addition, the cholesterol and apo B content of LDL were reduced significantly with a sparing effect on LDL α-tocopherol. This novel fibre preparation could be an effective diet therapy and therefore needs further investigation.     

Physiological Studies on Thiobacilli

Thiosulfate was oxidized to tetrathionate and an unknown oxidized sulfur compound. The activity was found in the particulate fraction (a precipitate obtained by centrifuging (at 90,000 × g for 60 min) the supernatant of centrifugation at 10,000 × g for 30 min). Treatment with deoxycholate caused inactivation of the enzyme. Considering from these points and electronmicrograph, the enzyme would be located on the cell membrane.     

Genetics of the peroxidase isoenzymes inPetunia

Summary The structural geneprxD inPetunia codes for a slow moving anodic peroxidase whose activity is sensitive to high concentrations of hydrogen peroxide. The PRXd enzyme could be found in mature and old leaf and stem tissue of full-grown flowering plants. PRXd was found to be absent in tissues from flower corolla and root. The geneprxD is the fourth gene that codes for peroxidases in leaf and stem. Two mobility variants of the PRXd enzyme have been found among our inbred lines using starch gel system II electrophoresis. The geneprxD could be located on chromosome III by a four-point-cross involving the genesprxA, prxD, Mf1 andHt1. The order of the genes established is:Ht1 — Mf1 — prxD — prxA.     

Iron chelators of the pyridoxal isonicotinoyl hydrazone class

Summary Formation constants for the calcium(II), magnesium(II) and zinc(II) complexes of the orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH) and three analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxalp-methoxybenzoyl hydrazone (PpMBH) and pyridoxalm-fluorobenzoyl hydrazone (PmFBH) have been determined by potentiometry at 25\dg C andI=0.1 M [KNO₃]. The four ligands bind calcium(II) weakly and magnesium(II) only slightly more strongly, as a l: l complex which is formed at pH \s> 8. The chelation of zinc(II) for all the ligands studied was greater than that for calcium(II) and magnesium(II), with complexation generally becoming significant at about pH 5. Thus, chelation of zinc(II) but not calcium(II) or magnesium(II) at physiological pH, 7.4 may be expected. Calculated values of the concentration of uncomplexed metal ion indicate that the selectivity of these ligands towards Fe(III) is comparable to that of the clinically used chelator desferrioxamine.