DESIGN OF PROCESS OF PRODUCTION OF AMYLASES FROM LIQUID OPTIMISED CULTURES OF ASPERGILLUS ORYZAE USING FLOUR MILL WASTE AS CARBON SOURCE.

BARRERA, VIRGINIA; CABEZUDO, IGNACIO; AGUILERA, JOEL A; ROMANINI, DIANA; BRAIA, MAURICIO

Guaruja, SP

Congreso; Biopartitioning & Purification Conference; 2019

Faculdade de Ciências Farmacêuticas - Câmpus de Araraquara UNESP

Valorisation of agricultural by-products is very important for developing sustainable economic and industrial activities. Agricultural wastes are hard to dispose, but they can be used to produce high-value biotechnological products such as enzymes, alcohols and organic acids. In particular, flour mill waste (FMW) is obtained during the production of wheat flour and is rich in starch (14-22 % dry weight), an excellent inducer for the expression of amylolytic enzymes in fungal fermentations and thus, they can be used as inductor to produce alpha-amylase (AMY) by liquid fermentation. The aim of this work was to design a production process of AMY from liquid optimised cultures using flour mill waste as only carbon source. Therefore, a design of experiments was prepared, considering a first stage of screening where it was found that the significant factors were FMW concentration, inoculum size, time and incubation temperature. Besides evaluating AMY production, since A. oryzae has the ability to secrete protease enzymes into the media, the incidence of factors on their production was also analysed, since their hydrolytic action, could tend to destabilize AMY extracts. The optimization of selected factors provided the best conditions for the production of α-amylase: 10 g/L of FMW, 100 conidia/mL inoculum, 28° C and 8 days of incubation. These values allowed for a maximum activity of 13.4 U AMY/L. Following production optimisation, purification and concentration of α-amylase took place in two stages: precipitation with (NH4)2SO4 was followed by hydrophobic interaction chromatography. Thus, the enzyme concentrated four times and purified eleven times, recovering 65% of its activity, while the proteolytic activity of the extract could be reduced to 14%. The purified α-amylase showed its maximum stability at 30 °C, in a pH range of 4-6; with its maximum activity at 35 °C, at pH 4.00. In this optimised process lays the possibility for the development of a production design on a larger scale of submerged cultures.