Centrifugal precipitation chromatography, a powerful technique for the isolation of active enzymes from tea leaves (Camellia sinensis)

Abstract

Centrifugal precipitation chromatography was developed approximately 10 years ago. In contrast to other counter-current chromatographic techniques, the centrifugal precipitation chromatography system is operated with two mutually miscible solutions separated by a cut-off membrane. Centrifugal precipitation chromatography was firstly introduced for the separation of proteins using an ammonium sulfate gradient. In this study we describe a novel approach using solvent-based protein precipitation for the isolation of active plant enzymes from tea leaves (Camellia sinensis) by centrifugal precipitation chromatography. We developed a gradient based on acetone and Tris-buffer, because the biological activity of carotenases in tea leaves cannot be preserved in the presence of ammonium sulfate. Parameters such as the critical solvent concentration, flow rate, buffer concentration, and sample load were determined and/or optimized. Subsequently, the newly developed separation protocol was successfully used for the isolation of active carotenoid cleavage enzymes from tea leaves. The isolated enzymes showed high enzymatic activities and purities and could be directly used for enzymatic assays and structure elucidation.

Figure 1

Enzymatic oxidation of β-carotene by specific carotenoid cleavage oxygenases in tea leaves

Figure 2

(a) Acetone concentrations used for protein precipitation in relation to protein content (measured by Bradford Assay) of the supernatant (unprecipitated proteins). (b) Carotenoid cleavage ability of a crude enzyme extracts obtained after precipitation with different acetone concentrations.

Figure 3

Osmosis rate and salt concentration in output fractions (sample channel) of the solvent system acetone-Tris-buffer, sample channel: Tris-buffer at a flow rate of 0.1 mL min^-1 and solvent channel: gradient of acetone-Tris-buffer 0-2 h 100 % acetone, 2-12 h 100 – 0 % acetone, 12-18 h 100 % Tris-buffer at a flow rate of 1 mL min^-1 and 1000 rpm.

Figure 4

Activity profile and calculated solvent gradient of a CPC separation (Separation conditions as given in the legend of Figure 2).

Figure 5

SDS-PAGE (NuPAGE 12 % Acrylamide) stained with SilverXpress Staining Kit. The numbering is equally to the CPC fraction number in Figure 3 - (A) 3,(B) 5,(C) 6,(D) marker, (E) 10, (F) 14, (G) 17.