Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent K m values for NADPH and Methyl Red substrates were 0?074 and 0?057 mM, respectively. The apparent V max was 0?4 mM min "1 (mg protein) "1 . Azo1was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora.

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An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celElike), and family 1 β-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA).Obligately anaerobic fungi are part of the natural microflora of the alimentary tracts of herbivores and they might account for up to 20% of the total microbial biomass in the rumen [4,17]. They have been isolated from digesta and feces of many herbivorous animals [17]. According to the number of sporangia developed from the thallus, they have been divided into monocentric and polycentric fungi [5]. Anaerobic fungi can colonize and penetrate plant tissues as a result of filamentous growth, which appears to degrade lignified tissue that is not degraded by other microorganisms in the rumen [13]. These fungi are active in the degradation of plant cell wall polysaccharides and represent a potential source of enzymes against cellulose and hemicelluloses. The importance of the anaerobic fungi is further supported by the synergism with rumen bacteria to enhance degradation of carbohydrates [13]. In addition, the anaerobic fungi may have the potential to contribute substantially to drug metabolism in the alimentary tract of host animals due to the ability to produce a wide range of enzymes with hydrolytic capacity [4]. Molecular evidence shows that hydrolytic Correspondence to: Huizhong Chen; huizhong.chen@fda.hhs.gov. High-throughput DNA sequencing and the advent of the proteomics disciplines now offer the potential to obtain a blueprint for the lifestyle of a specific microbe, and to access its genetic potential in a comparative and functional manner. The genome sequences of several rumen bacteria with relevance to fiber degradation, such as Fibrobacter succinogenes, Ruminococcus albus, and Prevotella ruminicola strains 23, are available [13]. Due to the highly AT-rich (80-85 mol%) genomes of anaerobic fungi and to the extent that these genomes test the limits of the physiochemical methods utilized in their analyses, relatively little information is available regarding the genomes of these fungi. Further hampering research are the low yields of DNA and the high carbohydrate contents normally associated with current isolation techniques. For example, the DNA content of 3-to 6-day-old cultures of Neocallimastix sp. is only 2.7-3.2 μg/mg dry weight or 0.32% by weight as determined by spectrofluorometric method on sonicated samples [1,5]. Subsequently, the biggest obs...