Produção de proteases neutras de cogumelos para aplicação na indústria de detergente

Abstract

Proteases are important commercial enzymes with several applications in industrial areas and it is an essential factor the search of proteases sources with new properties. For that reason, the aim of this study was to investigate the proteolytic activity of four mushrooms strains from DPUA Culture Collection and evaluate the regulatory mechanisms of the highest producer in order to industrial application. Auricularia mixotricha DPUA 1695, Ganoderma lucidum DPUA 1694, Lentinus citrinus DPUA 1693 and Pleurotus ostreatoroseus DPUA 1720 were inoculated in GYP medium [(2.0% (w/v) glucose + 1.0% (w/v) peptone + 0.5% (w/v) yeast extract)]. The parameters used to determine the influence in enzyme production were: carbon sources, nitrogen sources, inoculum size, cultivation time and medium volume. A 24 full factorial design, with 4 central points, was made to verify the influence of inducer substrate, inoculum age and initial pH. The determination of optimum pH was carried out with citrate (pH 4.0 to 6.0), phosphate (pH 6.0 to 8.0) and carbonate-sodium bicarbonate (pH 9.0 to 10.0) buffers; the optimum temperature in 30, 40, 50, 60, 70 and 80 ⁰C; and the effect of inhibitors was made using CaCl2, KCl, NaCl, CuSO4, FeSO4, MgSO4, ZnSO4, EDTA, PMSF, pepstatin and iodoacetic acid. The protease efficiency in removing stains was carried out using cotton cloths stained with acai pulp, cabbage juice and V8 vegetable juice® and the crude extract compatibility was compared to the commercial detergent brands Ace®, Ariel®, Minuano® and Omo®. P. ostreatoroseus was the selected strain presenting the highest protease production. According to the parameters evaluated, the cultivation using 5 discs of the mushroom mycelium in 40 mL of AYG [2.0% (w/v) starch, 0.5% (w/v) yeast extract + 1.0 % (w/v) gelatin] during 7 days promoted the highest protease production. In the factorial design, the highest activity was obtained in the medium containing 2.0 (w/v) starch + 0.5 % gelatin + 1.0 % yeast extract, pH 8.0 and inoculum of 16 days age. The proteases had maximum activity at pH 7.0 and 50 ⁰C. They were stable for 1 hour between pH 7.0 and 9.0 and for 30 minutes at 50 ⁰C. The metallic Fe ions stimulated the activity while K ions, EDTA, PMSF and iodoacetic acid inhibited the activity indicating the presence of metallic, serine and cysteine proteases. The crude extract was efficient in the removing of acai pulp stains and was compatible with the commercial detergent Minuano®. The enzymes characteristics, according to optimum pH and temperature, compatibility and stability, allow their utilization as additive in detergent industry.