Azoreductase activity

Ouwehand AC, Lagstrom H, Suomalainen T, Salminen S Effect of probiotics on constipation, fecal azoreductase activity and fecal mucin content in the elderly. Ann Nutr Metab 2002 46 159-162. 

Rafii F, Franklin W, Cemiglia CE (1990) Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora. Appl Env Microbiol 56 2146-2151... 

Dykes GA, Timm RG, von Floly A (1994) Azoreductase activity in bacteria associated with the greening of instant chocolate puddings. Appl Environ Microbiol 60 3027-3029... 

Reduction. The activity of azo- and nitroreductase varies between different species, as shown by the in vitro data in Table 5.11. Thus, azoreductase activity is particularly high in the guinea pig, relative to the other species studied, whereas nitroreductase activity is greatest in the mouse liver. 

Reductive reactions, like oxidation, are carried out at different rates by enzyme preparations from different species. Microsomes from mammalian liver are 18 times or more higher in azoreductase activity and more than 20 times higher in nitroreductase activity than those from fish liver. Although relatively inactive in nitroreductase, fish can reduce the nitro group of parathion, suggesting multiple forms of reductase enzymes. 

Azoreductase activity is present in both the endoplasmic reticulum and the cytosol of the liver. It is thought to produce the hydrazo intermediate that is cleaved to give two primary amines. The best known example is that of the azo dye Prontosil that is cleaved to form sulfanilamide, an active antibacterial compound, a reaction that involves a four-electron transfer. 

Azoreductase activity (substrate l,2-dimethyl-4-(/ -carboxyphenylazo)-5-hydroxybenzene (CPA) (Hanzel and Carlson 1974) and nitroreductase activity (substrate. 77-nitrobenzoic acid) (Carlson 1972) have been detected in digestive gland of M. mercenaria (Table 12). Their properties were similar to those of mammalian enzyme activities and considered indicative of the existence of two separate enzyme systems. Nitroreductase activity was maximal at 35 to 45 C and pH 6.0, Stimulated by flavin mononucleotide, and inhibited by potassium cyanide and air, but not by SKF-525A or carbon monoxide (cytochrome P-450 inhibitors). Azoreductase activity was similarly stimulated by flavin mononucleotide and inhibited by air, but had a pH optimum of 8.0 activity was higher at 37 C than at 22 °C. Both enzyme activities had cytosolic and microsomal subcellular localizations, viz. activity in nmol min g wet weight (% distribution in brackets)... 

Hansen LG, Kapoor IP, Metcalf RL (1972) Biochemistry of selective toxicity and biodegradability comparative (9-dealkylation by aquatic organisms. Comp Gen Pharmac 3 339-344 Hanzel ME, Carlson GP (1974) Azoreductase activity in the hard clam, Mercenaria mercenaria (L.). J Exp Mar Biol Ecol 14 225-229... 

Biological/Medical Applications Detecting risk of Alzheimer s disease and stroke evaluating/testing sperm quality identifying bacteria as a substrate for measuring aromatase activity, azoreductase activity, phospholipase activity, proteases activity (caspase activity, cathepsin C activity, elastase activity proteinase activity) " implantable drug-delivery devices ... 

Chevalier, A. Mercier, C. Saurel, L. Orenga, S. Renard, R Romieu, A. The first latent green fluorophores for the detection of azoreductase activity in bacterial cultures. Chem. Commun. 2013, 49, 8815-8817. 

Bafana A, Chakrabarti T, Devi SS (2008) Azoreductase and dye detoxification activities of Bacillus velezensis strain AB. Env Biotechnol 77 1139-1144... 

Liu G, Zhou J, Wang J (2009) Acceleration of azo dye decolorization by using quinone reductase activity of azoreductase and quinone redox mediator. Bioresour Technol 100 2791-2795... 

Based on the previous publications, azo dye can be reduced by azoreductase-catalyzed reduction under anaerobic conditions. But still there is a speculation whether bacterial flavin reductases are responsible for the azo reductase activity observed with bacterial cell extracts. In a published report, it is reported that flavin reductases are indeed able to act as azo reductases [24]. Bacteria produce extracellular oxidative enzymes, which are relatively nonspecific enzymes catalyzing the oxidation of a variety of dyes. It was reported that so many diverse groups of bacteria play a role in decolorization. It has been also reported that mixed microbial community could reduce various azo dyes, and members of the y-proteabacteria and sulfate reducing bacteria (SRB) were found to be prominent members of mixed bacterial population by using molecular methods to determine the microbial population dynamics [1],... 

The yeast-mediated enzymatic biodegradation of azo dyes can be accomplished either by reductive reactions or by oxidative reactions. In general, reductive reactions led to cleavage of azo dyes into aromatic amines, which are further mineralized by yeasts. Enzymes putatively involved in this process are NADH-dependent reductases [24] and an azoreductase [16], which is dependent on the extracellular activity of a component of the plasma membrane redox system, identified as a ferric reductase [19]. Recently, significant increase in the activities of NADH-dependent reductase and azoreductase was observed in the cells of Trichosporon beigelii obtained at the end of the decolorization process [25]. 

The metabolic transformation of many drugs is catalysed by various enzyme of the intestinal microflora. The anaerobic microflora and colon are rich in reductases which may be responsible for a significant proportion of the azoreductase and nitroreductase activity. The enzymes and other factors that may produce change in the nature of intestinal microflora might also produce changes in the metabolism pattern of the drugs. 

Sulfasalazine, balsalazide, and olsalazine contain 5-ASA bound by an azo (N=N) bond to an inert compound or to another 5-ASA molecule (Figure 62-8). In sulfasalazine, 5-ASA is bound to sulfapyridine in balsalazide, 5-ASA is bound to 4-aminobenzoyl-B-alanine and in olsalazine, two 5-ASA molecules are bound together. The azo structure markedly reduces absorption of the parent drug from the small intestine. In the terminal ileum and colon, resident bacteria cleave the azo bond by means of an azoreductase enzyme, releasing the active 5-ASA. Consequently, high concentrations of active drug are made available in the terminal ileum or colon. 

Table 5.11 Hepatic Azoreductase and Nitroreductase Activities of Various Species...

Alternatively, the enzyme in the gut can be utilized to control the release of the active drug in the gut. For example, sulfasalazine, which is employed in the treatment of ulcerative colitis, is a combination of sul-fapyridine and 5-aminosalicylate chemically linked via an azo bond. It remains absorbed and intact throughout the GI tract until it reaches the large intestine, where bacterial azoreductase enzymes degrade the azo bond and release sulfapyridine and 5-aminosalicylate to act locally on the lesions. 

TABLE 5.11 Hepatic azoreductase and nitroreductase activities of various species... 

An oligopeptide spacer with aromatic azo groups was used for selective release of 9-aminocamptothecin in colon. A combination of azoreductase and proteolytic activity was responsible for selective drug release in colon while the conjugate was stable in stomach and small intestine. Absorption of the drug in vivo after oral administration was also tested. ... 

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