Method to concentrate protein solutions based on dialysis–freezing–centrifugation: Enzyme applications

Abstract

One of the recurrent methodological problems in preparative biochemical work is the concentration of dilute protein solutions, including culture supernatants resulting from biotechnological processes. A procedure was developed to concentrate enzymes by a novel cryoconcentration system. This approach includes a new device that facilitates the sample freezing and the subsequent solute elution from the frozen matrix by centrifugation. The optimal centrifugation conditions for this cryoconcentration system were obtained using whey protein solution as a model. The procedure was applied to concentrate dilute solutions of commercial pectinase, measuring the endopolygalacturonase (EPG) activity of this enzyme in the concentrate by a method based on the on-line torque measurement, and of recombinant fructan:fructan 1-fructosyltransferase (1-FFT) protein of Pichia pastoris from a culture in a bioreactor, as an expression system. The optimal centrifugation speed, time, and temperature were 6150g, 20min, and 4°C, respectively. The concentration factors for the dilute protein solutions were 9.2-, 11.2-, and 17.1-fold for 1-FFT, whey, and commercial pectinase, respectively. Recoveries ranged from 87% to 93%. The procedure allowed concentrating proteins efficiently without affecting their enzymatic activity.