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

VIRGINIA LIS BARRERA; IGNACIO CABEZUDO; JOEL AGUILERA; DIANA ROMANINI; MAURICIO BRAIA

GUARUJÁ

Conferencia; BIOPARTITIONING & PURIFICATION CONFERENCE; 2019

UNIVERSIDAD ESTATAL PAULISTA (UNESP). SOCIEDAD BRASILERA DE MICROBIOLOGÍA

Valorisation of agricultural by-products is very important for developing sustainableeconomic and industrial activities. Agricultural wastes are hard to dispose, but they canbe used to produce high-value biotechnological products such as enzymes, alcoholsand organic acids. In particular, flour mill waste (FMW) is obtained during theproduction of wheat flour and is rich in starch (14-22 % dry weight), an excellentinducer for the expression of amylolytic enzymes in fungal fermentations and thus, theycan 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 optimisedcultures using flour mill waste as only carbon source. Therefore, a design ofexperiments was prepared, considering a first stage of screening where it was foundthat the significant factors were FMW concentration, inoculum size, time and incubationtemperature. Besides evaluating AMY production, since A. oryzae has the ability tosecrete protease enzymes into the media, the incidence of factors on their productionwas also analysed, since their hydrolytic action, could tend to destabilize AMY extracts.The optimization of selected factors provided the best conditions for the production ofα-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 productionoptimisation, 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%. Thepurified α-amylase showed its maximum stability at 30 °C, in a pH range of 4-6; with itsmaximum activity at 35 °C, at pH 4.00. In this optimised process lays the possibility forthe development of a production design on a larger scale of submerged cultures.