PRODUCTION AND PURIFICATION OF ALPHA-AMYLASE USING FUNGAL SOLID FERMENTATION OF TWO AGRO-INDUSTRIAL WASTES

CLEMENTZ, ADRIANA L.; MELNICHUK, NATASHA; MEINI, MARÍA-ROCÍO; GUERRA, LAUREANA; ROMANINI, DIANA

Guaruja, SP

Congreso; Biopartitioning & Purification Conference; 2019

Faculdade de Ciências Farmacêuticas - Câmpus de Araraquara UNESP

Valorization of agricultural by-products is very important for developing sustainable economic and industrial activities since they are produced in large quantities and many times, it is very difficult to properly dispose of them. In this way, agricultural wastes can be used to produce high-value biotechnological products such as enzymes, alcohols, and organic acids. Soybean and wheat are two of the most produced crops worldwide, being the USA, China, the UE, and Brazil and Argentina in South America, the major producers. Agricultural wastes such as soybean husk (SH) and flour mill waste (FMW) are produced during the processing of these crops. The first is obtained during the production of soybean oil and flour while the second is obtained during the production of wheat flour. Both are rich in starch (14-22 % dry weight) which is the best inducer for the expression of amylolytic enzymes in fungal fermentations and thus, they can be used as an inductor to produce alpha-amylase (AMY) by fermentation. The aim of this work was to employ solid-state fermentation using two wastes -soybean husk and flour mill waste- to produce and purify high quantities of alpha-amylase enzyme. The substrate composition and the culture conditions were assayed to improve alpha-amylase production by Aspergillus oryzae. The maximum productivity of the enzyme was achieved using a solid substrate composed of the two wastes, at 45 % soybean husk and 55 % flour mill by-product, without pre-treatment, at 30°C for 6 days. A very high alpha-amylase activity was yielded, 5,560 U/mL, which corresponds to 47,000 U/g dry substrate. The enzymatic extract obtained was characterized by LC-MS, providing a complete profile of the proteins produced during the solid-state fermentation on these two wastes. Then, the extract was purified by size-exclusion chromatography and the recovery and the purification factor of alpha-amylase enzyme were 83% and 6, respectively. Finally, the system was scaled up 50 times and yielded the same enzymatic activity.