Use of countercurrent chromatography applied on aqueous two-phase systems for purify bromelain from agroindustrial wastes

Abstract

Bromelain is the generic name given to all of the members derived endo-peptidases belonging to the Bromeliaceae family, which belongs to the pineapple (Ananas comosus). These enzymes represent an important class of enzymes with important physiological roles in a wide range of proven therapeutic applications (as anti-inflammatory, anti-tumor, adjuvant healing; increase in cardiorespiratory fitness, etc.) and, besides, are widely used commercially, being among the three largest groups of industrially used enzymes and responsible for 60% of the international enzymes sales. Aqueous Two-Phase Systems (ATPS) are simple, biocompatible, and very easy to scale up in operation, making their use very attractive for large-scale bioseparations. Countercurrent chromatography is a liquid-liquid partition chromatography process where both the mobile and stationary phases are liquids and the "column" is a long length of tubing which is wound on a drum that rotates in planetary motion. The permanent adsorption of the analyte onto the column, usual when using solid supports, is avoided and a near 100% recovery of the analyte can be achieved. Besides, the motion sets up a centrifugal force field that holds one liquid phase stationary against the flow of the other phase through zones of wave-like mixing and settling along the length of the column. Using a liquid stationary phases gives the process an increased sample loading capacity, ability to tolerate particles and a huge versatility as either phase can be mobile phase and there is no loss of sample as the retained stationary phase can simply be pumped out of the column at the end of a run. This project will report the feasibility of using countercurrent chromatography to purify bromelain from crude extract obtained from pineapple waste and as polishing step after its purification using aqueous two-phase systems. Therefore, we will be able to compare ATPS and CCC and determine which technique is more suitable to purify bromelain in the studied conditions. (AU)