Produção e aplicação de amilases por Aspergillus awamori: uma alternativa para a hidrólise enzimática de altas cargas de milho extrudado

Abstract

Corn ethanol production in Brazil could reach 8.2 billion liters in the 2024/2025 harvest and is based on the enzymatic hydrolysis of starch to obtain fermentable sugars. As an alternative to the conventional process, dry extrusion of corn favors starch breakdown and enzymatic hydrolysis under milder conditions. This study investigated the production of α-amylase and glucoamylase by Aspergillus awamori in different submerged culture media and its application in enzymatic hydrolysis with 30% and 40% solids. Six culture media were tested with different carbon and nitrogen sources. The best results were obtained with media M4 (5% commercial starch, 1.17% ammonium sulfate, and 0.1% yeast extract) and M6 (3% ground corn, 1% yeast extract, and 0.3% sodium nitrate), which showed the highest production of glucoamylase and alpha-amylase, respectively. The α-amylase:glucoamylase ratio ranged from 1.3:1 (M4) to 10.9:1 (M6), with M4 being selected for screening of the culture medium components via Plackett-Burman due to the ratio between amylases close to 1 being indicated for simultaneous liquefaction and saccharification. The design resulted in a 74% increase in enzyme production compared to the original M4 medium, reaching approximately 170 IU/mL of α-amylase and 140 IU/mL of glucoamylase, constituting the PB4 medium (7.5% commercial starch, 0.585% ammonium sulfate, and 0.15% yeast extract). Therefore, high glucoamylase activity and an α- amylase:glucoamylase ratio were achieved, allowing the study of simultaneous liquefaction and saccharification of extruded corn starch. Production in a 10 L bioreactor confirmed the feasibility of scaling up the process. In hydrolysis with enzyme loads of 66 IU and 131 IU α- amylase/g of biomass, similar yields were obtained in periods of 9 and 24 hours, indicating a potential reduction in enzyme use and time. The amylases maintained thermal stability up to 65 °C, with partial denaturation at 80 °C. Enzymatic hydrolysis was optimized using a central composite rotational design, evaluating the effects of pH and temperature for 24 h of reaction (200 rpm and a load of 66 IU/g of enzyme). Temperature was the main factor up to 6 h, while acidic pH favored stability after 9 h, leading to yields close to 80% after 24 h. The mathematical model obtained was validated experimentally, with an optimal yield of 82.34% ± 1.25 at pH 3.84 and 55.4 °C, statistically equivalent to the theoretical value (Student's t-test, 10%). The solid residue from hydrolysis contained 56% glucan and 12% oil, with the oil consisting mainly of linoleic acid (56%), indicating high economic potential in the recovery of hydrolysis coproducts.