“Produção, purificação, imobilização e parcial caracterização da lipase de Preussia africana”

Abstract

The present study sought to purify a new lipase produced by the endophytic fungus Preussia africana (P. africana). The enzymes were produced via submerged cultivation. After cultivation, the broth was filtered and freeze-dried so that the enzymes were concentrated. Organic solvents were used (methanol, 95% ethanol, absolute ethanol, iso-propanol and acetone), at temperatures of 10 and 25°C, for the precipitation of lipases, and it was observed that acetone and iso-propanol were more efficient. in precipitating the enzymes while maintaining their catalytic activity, obtaining specific activities of 211.34 ± 0.05 and 179.50 ± 0.08 U/mg, respectively. For these solvents, a new additional precipitation step was carried out in order to obtain greater recovered activity, varying the volume ratio between the supernatant resulting from the first precipitation step and the new solvent phase and the temperature (10 and 25°C). For the second precipitation step, a ratio of 1:3 supernatant: solvent (v/v) and a temperature of 10°C were used. A similar performance was obtained for both solvents, with a purification factor of 1.89 ± 0.01 and a recovered activity of 97.50% for iso-propanol and a purification factor of 1.88 ± 0. 01 and recovered activity of 96.70% for acetone. For the ratio of 1:2 supernatant: solvent (v/v) and 10 °C, for the second precipitation step, acetone showed better performance, obtaining a purification factor of 1.89 ± 0.01 and a recovered activity of 93.45%. After the precipitation steps, the enzymes were separated and the lipase with a molecular mass close to 45 kDa was purified and characterized according to the type of substrate, pH and temperature, so that the maximum hydrolytic activity observed was 72.80 + 1.29 U/mL, was obtained using olive oil as a substrate, at a pH between 7 and 8 at a temperature of 37°C. Butyl and octyl-agarose resins were used to evaluate the immobilization of the lipase obtained. The octyl-agarose resin showed a better immobilization yield, reaching 81.56%, however, the highest recovered enzymatic activity was obtained for the butyl-agarose resin (6.49%).