Purification and partial characterization of an exocellular proteinase fromTrichophyton rubrum

Abstract

An exocellular proteinase produced by Trichophyton rubrum in a glucose-peptone broth was purified from lyophilized and dialysed culture filtrate of the dermatophyte by Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified enzyme was a homogeneous protein of molecular weight 34 700 and it could hydrolyse azoalbumin, casein, bovine serum albumin, a-N-benzoyl-L-arginine ethyl ester and p-toluenesulfonyl-L-arginine methyl ester but not N-benzoyl-L-tyrosine ethyl ester, a-N-benzoyl-DL-arginine-p-nitroanilide and keratin. The enzyme showed an alkaline pH optimum and was not activated by divalent metal ions but inhibited strongly by phenylmethylsulfonylfluoride. Thus the enzyme was identified as an alkaline serine proteinase. Exocellular and mycelial proteinases occur commonly in dermatophytes of the genus Trichophyton, including T. rubrum. These enzymes could play a prominent role not only in growth and multiplication (ensuring nutrient supply from the proteinaceous components of the skin, hair or nail) but also in the infection of the host tissue. Minocha et al. [18] reported on the role of proteolytic enzymes in the manifestation of inflammatory reactions in hosts with dermatophyte infections. Exo-or endocellular proteinases have been purified to homogeneity for detailed enzymatic studies from different dermatophytes, e.g.T, granulosum [8], T. mentagrophytes [32,33,34], Microsporum gypseum [11,24] and M. canis [25].Several groups of workers demonstrated the presence of proteinases and peptidases in the cells or culture filtrate of T. rubrum [2,3,4,17,26]. The exo-and endocellular proteinase activity of this fungus showed multiple pH optima and probably represented a mixture of closely related enzymes. Accumulation of proteolytic activity in the culture filtrate of T. rubrum was stimulated if keratin was added as growth substrate [2,17] and suppressed by glucose, various carbohydrates and amino acids [17]. However, no successful attempts were made for the purification of these enzymes from T. rubrum though a limited degree of purification has been reported by Chattaway et al. [2] and Meevootisom & Niederpruem [17].

Secreted subtilisin gene family in Trichophyton rubrum

“…Two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T. rubrum grown in a medium containing soy protein as a sole nitrogen source. With an optimum pH of activity between 8.0 and 9.0, the previously isolated T. rubrum 34.7 kDa serine alkaline protease (Sanyal et al, 1985) and serine keratinolytic proteases active as dimers with subunits of 36 or 44 kDa (Asahi et al, 1989) may be Sub3 for which the molecular mass was estimated as 33 kDa on SDS-PAGE. Recombinant Sub3 and Sub4 were more active on keratin azure in presence of h-mercaptoethanol.…”

“…Although secreted metalloprotease activity was rather dominant, the serine protease activity was about 25 -50% of the total activity of the culture supernatant as attested by inhibition of this fraction by PMSF. One 34.7 kDa serine alkaline protease (Sanyal et al, 1985) and two serine keratinolytic proteases with molecular masses of 93 and 71 kDa (Asahi et al, 1989) were isolated and characterized in T. rubrum. These proteases were shown to be active as dimers with subunits of 44 and 36 kDa, respectively.…”

Isolation and properties of extracellular proteinases from Sporothrix schenckii

“…2 and 6). Coexistence of more than one extracellular proteinase has also been reported for T. rubrum (3,12,18), Trichophyton mentagrophytes (27,28), and C. albicans (16,17).…”

Purification and characterization of an extracellular proteinase from hendersonula toruloidea

“…In dermatophyte and fungal infections, there is clear evidence suggesting that proteinases produced by these organisms play an important role in the pathogenesis of tissue invasion (2,11,21,25,(28)(29)(30)(31). Although H. toruloidea causes a similar infection, no proteinase from this organism has been reported.…”