Produção de esterases por Aureobasidium pullulans URM 7059 utilizando glicerol residual

Abstract

Exploring the microbial biodiversity opens several possibilities regarding the discovery of new enzymes with biochemical advantages over the existing ones that are potentially useful for many industrial applications. The esterases (E.C. 3.1.1.1) belong to the class of carboxyl ester hydrolases that catalyze the cleavage of triglyceride ester bonds. Thus, in the current work, the esterase production by Aureobasidum pullulans URM 7059, using the residual biodiesel glycerol as a carbon source, was optimized. The highest esterase activity was obtained in a rotatory shaker using a medium composed of glycerol (0.1% v/v), (NH 4 ) 2 SO 4 (4 g/L) and yeast extract (8 g/L). The enzyme was partially purified and its molar mass (50 kDa) was estimated by SDS-PAGE and zymography. The enzyme is stable at pH 6.0 to 7.0 in 90 % and temperatures below 30°C. Inorganic salts caused a pronounced decrease in the enzyme activity. Cu 2+ and Al 3+ did not affect the esterase activity, while Ca 2+ promoted the highest activity loss. The kinetic parameters K m and V max measured were 1.4 mM and 218 µmol min -1 (p-NPC) and 1.55 mM and 76.76 µmol min -1 (p-NPB), respectively. Ultimately, these values showed that the enzyme partially purified is an esterase due to the action in short –chain p- nitrophenol esters. The enzyme was produced in batch and airlift reactor. Then, studies were carried out to increase the scale of production in a stirred tank and airlift bioreactors. The activity values in stirred tank reactor (2L/min) were 18.3 ± 0.9 U/mL (p-NPC) and 27 ± 1.3 U/mL (p-NPB). Tests with different aeration rates (2, 4, 6 and

8L/min) in airlift reactors suggested that the maximum activities are related to the higher oxygen flow rate. The enzyme activity obtained at 8 L of air/min (airlift), was 35 U/mL (culture broth) at 24 hours of fermentation. The kinetic parameters K m and V max measured were 1.4 mM and 218 µmol min -1 (p-NPC) and 1.55 mM and 76.76 µmol min - 1 (p-NPB), respectively. The enzyme was precipitated in NH 4 SO 4 . The esterase activity bands in native gel revealed proteins with molecular mass of 172 kDa, 66 kDa and 40 kDa. The esterase produced by A. pullulans URM 7059 in airlift bioreactor was also able to degrade the MACO-St biopolymer with stability in wide range of pH (7.0 – 9.0) and temperature (40 ºC – 80 ºC).