Proteases de leveduras para aplicação médica

Abstract

The proteases are enzymes which occupy a pivotal position with respect to their application in medical and commercial fields. Collagenases are detached among several kind of proteases, used in pharmaceutical and cosmetician industry, in processes such as smooth the skin, oral hygiene, disinfect suppurative wounds, cicatrizing processes, burning and as a trombolytic agent. In order to select protease producing yeasts, this study had as objectives to partially characterize the enzyme as for pH and stability temperature and to verify the effect of the enzyme in the degradation of collagen. 50 samples of yeasts isolated from different substrates, obtained from the culture collection DPUA, were analysed. Samples were cultured in Malte extract supplemented with 1,0 % gelatin at 30 ºC in shaking conditions (140 rpm). The qualitative proteolytic activity was determined in agar gelatin-milk medium and the quantitative activity was determined using azocasein 1,0 % as substrate. The best culture conditions to protease production were established for a species, being studied the following parameters: characteristics of pathogenicity, size and age of inoculate, best growth temperature, coal and nitrogen sources. The best stability conditions of protease were assessed for pH (6-12) and temperature (25 º, 37 º, 40 º, 50 º, 60 º, 70 º and 80 ºC) and the effect of the enzyme in the degradation of collagen. Among the identified species, Trichosporon pullulans was selected, because it showed the highest levels of proteolytic activity in qualitative (halo = 27 mm) and quantitative (420 U/mL) assays. The assays to determine the characteristics of pathogenicity of this species showed positive growth at 37ºC, weak positive ureasic activity and there was no phospholipasic activity, the highest proteolytic activity (246,66 U/mL) detected with inoculate corresponded to 10% of the volume of medium and stock of culture growth for 24 hours. Trials to verify the effect of temperature (25 ºC to 50 ºC) on growth and protease production demonstrated that the higher biomass (3,46 mg/mL) and proteolytic activity (513,33 U/mL) were obtained at 30 ºC. Sucrose 1,0 % was the best coal source yielding higher biomass (6,18 mg/mL) and proteasic activity (580 U/mL) and as nitrogen source detached peptone 0,5 % showing the higher values of proteolytic activity (755,55 U/mL) and biomass (7,55 mg/mL). The collagenolytic activity was determined in solid medium using dissolvable collagen as the only coal source. T. pullulans degraded collagen, producing a 28 mm halo. This work demonstrated that protease, with collagenolytic activity; produced by T. pullulans, can be used as therapeutic agent further detailed studies.