The detection and quantification of novel non-haem extracellular glycosylated peroxidases produced by the thermophilic actinomycete Thermomonospora fusca BD25 by means of PAGE-zymogram.

Polyacrylamide gel electrophoresis (PAGE) is a common technique used in Biochemistry for separation of macromolecules [I]. It is particularly useful to ascertain the degree of protein purity and confirm molecular masses obtained from chromatographic methods [2-51. In addition PAGE can be developed as zymogram (native gel) to detect the presence of enzyme activity [6]. In this report we describe the condition for developing PAGE-zymogram to confirm the presence of non-haem peroxidase in extracellular supernatant produced by the thermophilic actinomycete Thermomonospora fuscu BD25 [7-81 during growth in a minimal salt medium containing oat-spelts xylan as the primary carbon source. The PAGE is developed as zymogram in the presence of a range of substrates. The substrate specificity of this enzyme will be compared to the extracellular peroxidases produced by other actinomycetes such as Steptomyces thermovioluceus [9]. In addition, the use of PAGEzymogram, demonstrates the true nature of the non-haem peroxidase and this novel feature will be illustrated. Polyacrylamide native gels (10% w/v) containing peroxidase samples were incubated with a range of substrates: L-3,4-dihydroxyphenylalanine (L-DOPA), caffeic acid, procatechuic acid and 2,4-dichlorophenol (2,4-DCP). Gels were flooded with 50 mM H202 in 0.1 M potassium phosphate buffer (pH 7.0) at room temperature in the presence of 10 mM solution of the substrates containing 0.05 M 4-aminoantipyrine (except for L-DOPA ). The gels were also developed with 3,3’-diaminobenzidine, 4-chloro-l-naphthol, o-dianisidine and 2,2’-azino-di-[3ethyl-benzthiazoline-(6)-sulphonic acid] (ABTS) containing 1 ml substrate solution (10% substrates dissolved in ethanol) mixed with 8 ml of 0.1 M sodium phosphate buffer (pH 6.0) and 1 ml of 30 mM H 2 0 2 . When the stained peroxidase bands appeared on the gels, they were photographed immediately.