Optimization of Lactoperoxidase and Lactoferrin Separation on an Ion-Exchange Chromatography Step

Abstract

Lactoperoxidase (LP), which is a high-value minor whey protein, has recently drawn extensive attention from research scientists and industry due to its multiplicity of function and potential therapeutic applications. In this study, the separation and optimization of two similar-sized proteins, LP and lactoferrin (LF) were investigated using strong cation exchange column chromatography. A two-step optimization strategy was developed for the separation of LP and LF. Optimization was started with central composite design-based experiments to characterize the influences of different decision variables, namely, flow rate, length of gradient, and final salt concentration in the linear elution gradient step on the yield of LP. This was followed by a more accurate optimization of ion-exchange chromatography (IEC) separation of LP and LF based on an experimentally verified chromatographic model. The optimal operating points were found and the results were compared with validation experiments. Predictions respecting yield confirmed a very good agreement with experimental results with improved product purity.

The Multifaceted Roles of Bovine Lactoferrin: Molecular Structure, Isolation Methods, Analytical Characteristics, and Biological Properties

“…flow rate of the mobile phase, buffer pH, and ionic strength. 85 Uchida pointed out that the efficiency of separation of LF from LPO might be improved by use of a buffer solution with pH >5 and ionic strength higher than 0.5. 86 For instance, Ng et al applied two-step isocratic elution with 0.4 and 0.6 M sodium chloride buffer (pH 7) for fractionation of an LF−LPO mixture.…”

“…The problem of LPO elimination could be solved by selection of the appropriate isolation conditions, e.g. flow rate of the mobile phase, buffer pH, and ionic strength . Uchida pointed out that the efficiency of separation of LF from LPO might be improved by use of a buffer solution with pH >5 and ionic strength higher than 0.5 .…”

Heme Peroxidases at Unperturbed and Inflamed Mucous Surfaces

“…At neutral pH values, all of the three heme peroxidases are cationic proteins. The isoelectric point of LPO is about 9.5 [ 41 ]. The corresponding values for the two other peroxidases are usually given as >10 for MPO [ 42 ] or >11 in the case of EPO [ 43 ].…”

Effect of Geometrical Parameters on Extraction Efficiency of the Annular Centrifugal Contactor

“…Kadam et al [17] had given the mass transfer coefficient (k c a) of the centrifugal extraction as a function of P/V, as shown in Equation (6). k c a = 0.64(P/V) 0.22 (6)…”

“…RSM uses a multivariate quadratic function (MQF) to represent the relationship between variables and response, while the interaction term coefficients characterize the degree of interaction between variables. RSM is widely applied to study extraction processes for its intuitive response surface plots [16,17]. The analysis steps of RSM can be referenced in Silva's work [18].…”