characterization of serine-proteases from P. hypophthalmus epithelial mucus as a potential feedstock for biocosmetic applications

Abstract

Chemical peeling is a cosmetical treatment that promotes skin renewal, by the remotion of skin layers through the appliance of corrosive compounds. It has proven to be successful for the removal of acne, scars, photoaging, and pigmentary lesions. Yet this procedure is aggressive and can produce several complications, among them infections, eruptions, erythema or scarring. As an alternative, enzymatic peelings have been proposed. Enzymes lead natural desquamation processes, principally by serine proteases (SP). SP have also been identified in fish epithelial mucus. As well, empirical evidence has shown that direct contact with Iridescent shark (Pangasius hypophthalmus) epithelial mucus, promotes skin regeneration. Hence, through the present study, the characterization of the proteases present in P. hypophthalmus epithelial mucus was held, in order to identify new SP with potential cosmetical use. Epithelial mucus was extracted by rinsing specimens in extraction buffer (NaCl 50mM pH 7.4) in polyethylene bags and held back to tank. The obtained extracts were pooled and centrifuged. Supernatant was concentrated and desalted using vacuum evaporation and PD-10 columns. Protease activity was evaluated through caseinolytic activity and zymography using casein and gelatin universal protease substrates. Also in-gel inhibition (PMSF, benzamidine, EDTA, O-phenanthroline, and iodoacetamide) and activation (Zn2+ , Ca2+ , K+ , Na+ and no ion) for specific protease families were evaluated. Caseinolytic activity was detected (5.33 ± 0.37 U/mg at 25 oC). As for zymography, active bands within 130-15 kDa were identified for gelatin, while only one active band of 63 kDa was identified for casein. Compared to control treatment (Zn2+), K+ and Na+ enhanced gelatinolytic activity of medium weight bands (63, 58 and 48 kDa), while Ca2+ depleted most protease activity. Serine and cysteine protease inhibitors, PMSF and iodoacetamide, excerpted similar inhibition by reducing 63 kDa and inhibiting 58, 56, 30 kDa activity. MMP inhibitors exerted slight inhibition to superior weight bands (114, 90 and 71 kDa). Benzamidine only depleted 45 kDa activity. The present study proves the presence of the MMPs and SPs within P. hypophthalmus epithelial mucus. This positive result opens the possibility for further protease characterization and isolation for their evaluation as feasible agents for biocosmetic treatments.