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Antimicrobial properties of porphyrins.

A large number of natural and synthetic porphyrins of diverse chemical compositions and characteristics can be isolated from nature or synthesised in the laboratory. Antimicrobial and antiviral activities of porphyrins are based on their ability to catalyse peroxidase and oxidase reactions, absorb photons and generate reactive oxygen species (ROS) and partition into lipids of bacterial membranes. Light-dependent, photodynamic activity of natural and synthetic porphyrins and pthalocyanines against Gram-positive and Gram-negative bacteria has been well demonstrated. Some non-iron metalloporphyrins (MPs) possess a powerful light-independent antimicrobial activity that is based on the ability of these compounds to increase the sensitivity of bacteria to ROS or directly produce ROS. MPs mimic haem in their molecular structure and are actively accumulated by bacteria via high affinity haem-uptake systems. The same uptake systems can be used to deliver antibiotic-porphyrin and antibacterial peptide-porphyrin conjugates. Haemin, the most well known natural porphyrin, possesses a significant antibacterial activity that is augmented by the presence of physiological concentrations of hydrogen peroxide or a reducing agent. Natural and synthetic porphyrins have relatively low toxicity in vitro and in vivo. The ability for numerous chemical modifications and the large number of different mechanisms by which porphyrins affect microbial and viral pathogens place porphyrins into a group of compounds with an outstanding potential for discovery of novel agents, procedures and materials active against pathogenic microorganisms.

Anti-Bacterial Agents↗

Droplet-based micro-flow chemiluminescence system for in vivo glucose determination by microdialysis sampling.

A micro-flow chemiluminescence (CL) system in vivo for glucose determination by the on-line microdialysis sampling is described in this paper. The micro-flow CL system uses discrete sample droplets, which formed at the tip of the capillary with the sampling volume of 4.5 microl. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and glucose oxidase (GOD) on the inside surface of the micro-flow cell which was fabricated in polymethyl methacrylate (PMMA). The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and H2O2, then H2O2 oxidizing luminol to produce CL in presence of HRP. The microdialysis probe was utilized for sampling in the rabbit blood; the sample throughput was 20 h(-1). The glucose level in blood of the rabbit was on-line monitored with good results.

Animals↗

Recent advances in azo dye degrading enzyme research.

Azo dyes, which are characterized by one or more azo bonds, are a predominant class of colorants used in tattooing, cosmetics, foods, and consumer products. These dyes are mainly metabolized by bacteria to colorless aromatic amines, some of which are carcinogenic, by azoreductases that catalyze a NAD(P)H-dependent reduction. The resulting amines are further degraded aerobically by bacteria. Some bacteria have the ability to degrade azo dyes both aerobically and anaerobically. Plant-degrading white rot fungi can break down azo dyes by utilizing a number of oxidases and peroxidases as well. In yeast, a ferric reductase system participates in the extracellular reduction of azo dyes. Recently, two types of azoreductases have been discovered in bacteria. The first class of azoreductases is monomeric flavin-free enzymes containing a putative NAD(P)H binding motif at their N-termini; the second class is polymeric flavin dependent enzymes which are studied more extensively. Azoreductases from bacteria represent novel families of enzymes with little similarity to other reductases. Dissociation and reconstitution of the flavin dependent azoreductases demonstrate that the non-covalent bound flavin prosthetic group is required for the enzymatic functions. In this review, structures and carcinogenicity of azo colorants, protein structure, enzymatic function, and substrate specificity, as well as application of the azo dyes and azoreductases will be discussed.

Animals↗

2,2'-Azo-bis-amidinopropane as a radical source for lipid peroxidation and enzyme inactivation studies.

1. 2,2'-Azo-bis-amidinopropane (ABAP) thermal decomposition produces free radicals that initiate the lipid peroxidation of erythrocyte ghost membranes. 2. Addition of 6-n-propyl-2-thiouracil decreases the rate of the process, both by decreasing consumption of the natural antioxidants of the membranes and by direct interaction with the free radicals involved in the lipid peroxidation. 3. Peroxyl radicals produced in ABAP thermal decomposition inactivate lysozyme, horseradish peroxidase (HRP) and glucose oxidase, in that order. The number of enzyme molecules inactivated per radical introduced into the system increases with enzyme concentration. 4. Competitive studies employing mixtures of enzymes show that the order of reactivity of these enzymes towards the peroxyl radicals is the opposite to that obtained for the rate of enzyme inactivation. It is concluded that inactivation efficiency is determined mainly by the average number of free radicals that must react with an enzyme molecule to produce its inactivation, and that this number is directly related to the molecular weight of the enzyme.

Amidines↗

Novel nitric oxide-liberating heme proteins from the saliva of bloodsucking insects.

The spectroscopic (UV-visible, IR, RR, MCD, Mössbauer, EPR), crystallographic, kinetic, and redox investigations that have been carried out on model hemes, hemoglobin, myoglobin, cytochrome a3 of cytochrome oxidase, horseradish peroxidase, prostaglandin H synthase, cytochromes P450, chloroperoxidase, and so forth have shown us the unique properties of heme-NO centers, as summarized above. However, in none of these cases is the Fe(III)NO complex of any known physiological importance. The nitrophorins of R. prolixus [59] (and Cimex lectularius [80]) are thus far unique in this respect. It is likely that further investigations of the roles of NO in biological systems will discover additional interesting involvements of heme proteins in these roles.

Amino Acid Sequence↗

[Enzymes of white rot fungi involved in lignin degradation].

The presence of oxidases and peroxidases was tested qualitatively in 12 strains of white rot Basidiomycetes. Plate tests with gallic acid, tannic acid, guayacol, Poly R-478 and Azure B were used. Fomes sclerodermeus, Phlebia sp. and Pycnoporus sanguineus were selected for further studies because they produced the largest areas of degradation in all media tested. Poly R-478 degradation and manganese peroxidase, lignin peroxidase and laccase activities were measured in glucose-asparagine (N-sufficient) and Kirk (N-limited) media. The highest activities were produced by F. sclerodermeus cultured in glucose asparagine medium.

Culture Media↗

Importance of enzyme purity and activity in the measurement of total dietary fiber and dietary fiber components.

A study was made of the effect of the activity and purity of enzymes in the assay of total dietary fiber (AOAC Method 985.29) and specific dietary fiber components: resistant starch, fructan, and beta-glucan. In the measurement of total dietary fiber content of resistant starch samples, the concentration of alpha-amylase is critical; however, variations in the level of amyloglucosidase have little effect. Contamination of amyloglucosidase preparations with cellulase can result in significant underestimation of dietary fiber values for samples containing beta-glucan. Pure beta-glucan and cellulase purified from Aspergillus niger amyloglucosidase preparations were used to determine acceptable critical levels of contamination. Sucrose, which interferes with the measurement of inulin and fructooligosaccharides in plant materials and food products, must be removed by hydrolysis of the sucrose to glucose and fructose with a specific enzyme (sucrase) followed by borohydride reduction of the free sugars. Unlike invertase, sucrase has no action on low degree of polymerization (DP) fructooligosaccharides, such as kestose or kestotetraose. Fructan is hydrolyzed to fructose and glucose by the combined action of highly purified exo- and endo-inulinases, and these sugars are measured by the p-hydroxybenzoic acid hydrazide reducing sugar method. Specific measurement of beta-glucan in cereal flour and food extracts requires the use of highly purified endo-1,3:1,4 beta-glucanase and A. niger beta-glucosidase. Beta-glucosidase from almonds does not completely hydrolyze mixed linkage beta-glucooligosaccharides from barley or oat beta-glucan. Contamination of these enzymes with starch, maltosaccharide, or sucrose-hydrolyzing enzymes results in production of free glucose from a source other than beta-glucan, and thus an overestimation of beta-glucan content. The glucose oxidase and peroxidase used in the glucose determination reagent must be essentially devoid of catalase and alpha- and beta-glucosidase.

Aspergillus niger↗

[Photometric method for determining ethanol].

A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.

Alcohol Oxidoreductases↗

[Characterization of inductive synthesis of levoglucosan kinase by a combined strategy of enzymological and fast atom bombardment mass spectrometric analysis].

Levoglucosan is the main product derived from pyrolysis of cellulose. A mutant Aspergillus niger CBX-209 could grow on levoglucosan well fermenting it into citric acid with a yield comparable to that on glucose. Levoglucosan hydrolase was absent by measuring glucose formation with the glucose oxidase and peroxidase coupling system. Cell extracts were partly purified by ammonium sulfate fractionation and ion-exchange chromatograph. Direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl2 was observed when it was reacted with partly purified enzyme by a combined strategy of enzymological and fast atom bombardment mass spectrometric analysis. These data showed that the mutant used a novel enzyme, levoglucosan kinase, to convert levoglucosan into glucose 6-phosphate. Levoglucosan kinase was an inductive enzyme.

Aspergillus niger↗