Lactoperoxidase purification from whey by using dye affinity chromatography

Abstract

Bovine lactoperoxidase is a glycoprotein present in milk, whey and colostrum, which might be used in dairy, cosmetic, pharmaceutical, veterinary and agricultural applications due to its broad antimicrobial activity. Here, we describe a novel process for bovine lactoperoxidase purification by using dye affinity chromatography. Eighteen triazine dyes were immobilized on Sepharose 6B and screened for their performance as possible ligands. Five of the dye-Sepharose matrices showed over 90% adsorption of bovine lactoperoxidase directly from whey without any pretreatment using the batch mode, and were thus selected for further adsorption and elution studies. The highest elution degree was obtained using 20 mM acetate buffer, pH 5.0, 2 M NaCl, as the eluent for all the matrices. Whey processed using the Reactive Red 4-Sepharose matrix in batch mode showed the highest bovine lactoperoxidase purification yield (86.5 ± 3.8%), purification factor (46.1 ± 1.1), and a relative purity higher than 80% according to SDS-PAGE gel densitometry. Whey processed using packed-bed column mode showed lower yields and additional whey pretreatments were needed for dynamic processing. The interaction between bovine lactoperoxidase and Reactive Red 4-Sepharose matrix was characterized using Langmuir isotherm model. The Kd value was 0.21 ± 0.03 mg/mL and the Qmax was 32.21 ± 1.24 mg/g. The results presented here suggest the potential application of the Reactive Red 4-Sepharose matrix to one-step purification of bovine lactoperoxidase from whey.