Evaluation of Column Flotation in the Downstream Processing of Fermentation Products: Recovery of a Genetically Engineered α‐Amylase

Abstract

Flotation is a simple, inexpensive, and versatile unit operation with a largely unexplored potential in biotechnology. There is a general lack of research concerning biotechnological applications in this area, especially in the recovery of fermentation products. Moreover, the few reports in the literature do not consider the modern concept of column flotation as practiced in the mineral industry. We report herein the application of column flotation for the recovery of a Bacillus stearothermophilus alpha-amylase expressed in Escherichia coli by the use of a food-grade polymer, (hydroxypropyl)methylcellulose (HPMC), and ammonium sulfate. First, the enzyme was removed from the liquid phase by partition to a salted-out HPMC phase. The enzyme-containing polymer flocs were then floated from the liquid. Recovery of active enzyme was as high as 90%, with throughput as high as 94 m3/(h.m2). The floatability of the enzyme from a periplasmic extract was higher than extracellular enzyme in the broth due to the presence of depressors of molecular weight lower than 10,000 in the broth.

Enzyme Production

“…This technique has also the potential for protein fractionation (Sarkar et al 1987;Miranda and Berglund 1993;Varley et al 1996;Brown et al 1999a,b), but this has not been fully appreciated, maybe because yields of recovery are low due to enzyme inactivation at the gas-liquid interface. The most relevant operation for enzyme concentration is ultrafiltration.…”

Effective Purification Procedure of Aspergillus Oryzae ?-Amylase From Solid State Fermentation Cultures Including Concanavalin a-Sepharose

“…Several purification methods are reported in the literature for amylolytic enzymes (Turchi and Becker 1987;Kong et al 1991;Rozie et d . 1991;Brandani et al 1993;Miranda and Berglund 1993), However, although Aspergillus oryzae a-amylase is a glycoprotein of the high mannose type (Yamaguchi et ul. 1971), none of these protocols includes the use of the lectin concanavalin A (ConA), which has high affinity for this class of glycoproteins.…”

Avaliação do efeito do sal volatil carbamato de amonio sobre a estabilidade de enzimas

“…É de grande interesse quando se consegue remover proteases que comprometeriam a integridade da molécula alvo (produto). Como citado no caso de precipitação isoelétrica, é fundamental que esta molécula alvo seja termoestável na temperatura de precipitação (MIRANDA e BERGLUND, 1993).…”