Phosphate-Mediated Alteration of theMicrosporum gypseumGermination Protease Specificity for Substrate: Enhanced Keratinase Activity

Abstract

Inorganic phosphate was found to decrease the caseinolytic and ethyl-esterase activities of the Microsporum gypseum germination protease. The germination protease possessed exokeratinase (beta-keratinase) activity immediately after release from the fungal spore. After phosphate treatment of the enzyme, the germination protease also possessed endo-keratinase (alpha-keratinase) activity. Phosphate altered the protease's pH optimum from 9.0 to 7.0 and decreased the molecular weight from 33,000 to 16,000. These values were identical to those found for the keratinase. Alphaand beta-keratinase activities were stimulated in excess of 200-fold by disulfide reducing agents. Natural and suspected keratin degradation products also enhanced keratinase activity. Cell fractionation and in vitro conversion of the alkaline germination protease into a functional keratinase suggested that the subunits comprising the germination protease and the keratinase were of a common origin.

Effect of reducing agents on proteolytic and keratinolytic activity of enzymes of Microsporum gypseum

“…The data comparable with our results are still sporadical in the literature. Page & Stock [29] studied a protease from M . gypseum macroconidia and recorded a 129-up to 790-fold increase in 'keratinase' activity in the presence of millimolar concentrations of sulphite, cysteine and dithiothreitol.…”

Extracellular protease expression in Microsporum gypseum complex, its regulation and keratinolytic potential

“…The presence of proteolytic enzymes in preparations from different dermatophytes and other pathogenic fungi have been reported by various workers. [16][17][18][19][20][21][22][23][24][25][26][27][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] While it has been suggested that strain differences reflect differences in proteolytic activities of pathogenic fungi, we know of no studies comparable with ours which demonstrate fundamental homologies in the proteolytic systems of different strains within a single taxon, although the proteases they produce may have adaptive specificities related to the substrates and the tissues that they invade. The homologies in the quantitative composition of proteases of two isolates of M. gypseum emerging from the same ecological group (soil) in this study are explicable in epidemiological terms corresponding to reported multifold protease interstrain variability between clinical and environmental isolates of Microsporum canis and Aspergillus niger.…”

“…As a result of the proximity of M. gypseum to superficial dermis layers, skin was the substrate of choice not employed in most of the previous studies on dermatophyte keratinolytic activity determinations. [24][25][26][27]31,32 Degradation of buffalo skin by M. gypseum cultures was connected with the synthesis of proteolytic enzymes released extracellularly. Another noteworthy index for measuring M. gypseum keratinolytic activity appeared to be gravitationally measurable loss of keratin substrate coupled with mycelial yield (biomass) of the fungus at the expense of degraded products released into the medium.…”

“…A few of the sporadic reports on M. gypseum protease activity dealt with the studies on regulation of enzyme activity in the fungus. [24][25][26][27] To date, the extent of variability in the type and quantity of proteolytic enzymes produced by dermatophytes is an issue that have received minimum of attention. The present communication therefore, is a report on the expression and regulation of extracellular proteases in two strains of M. gypseum complex with their role in the degradation of buffalo skin.…”

“…Determination of keratinolytic activity of a fungus in vitro would imply that keratinases have significant implications in the invasion of host tissues in vivo and their regulation would appear to be a major step towards a better understanding of the pathogenicity of the fungus and the host–parasite relationships and therefore in evolving new treatment strategies. A few of the sporadic reports on M. gypseum protease activity dealt with the studies on regulation of enzyme activity in the fungus 24–27 . To date, the extent of variability in the type and quantity of proteolytic enzymes produced by dermatophytes is an issue that have received minimum of attention.…”