Abstract
An approach to describe continuous partitioning of Cerrena unicolor laccase in a PEG 6000—phosphate aqueous two-phase system was proposed. The laccase was separated from crude supernatant of C. unicolor-submerged culture, and all the experiments were carried out in 25 °C and pH 7 conditions. Masses of both phases and their compositions at phase equilibrium, as well as laccase activity concentrations at different mixing points, were measured in batch experiments. An empirical short-cut method was developed which allows for calculation of mass and volume fractions of the phases, laccase concentration factors, and laccase recoveries. Theoretical predictions were verified by several experiments carried out in a special mixer-settler unit with automatic substrate feed and continuous collection of separated phases. Required concentration of the laccase was possible to achieve in a one-step extraction process in the mixer-settler unit. The predictions of the short-cut method were compared to the results of experimental measurements of phase compositions, phase volume fractions, concentration factors and enzymatic yields at steady-state operation of the extraction unit. The values of experimental results lay well within the 10% error range of the predicted values.
Highlights
Laccases (EC 1.10.3.2, p-diphenolic oxidases) are extracellular enzymes belonging to the oxidoreductase group, first discovered by Yoshida (1883)
The scope of this paper is to present a short-cut method of continuous extraction of Cerrena unicolor laccase with a PEG 6000—phosphate aqueous two-phase system, based on data from batch-extraction experiments
Mixing points corresponding to low-bottom phase volume fractions (Mixtures with low relative content of phosphate, such as experiment nos. 18, 22, 25) gave relatively high concentration factors because the majority of the enzyme was concentrated within a small volume
Summary
Laccases (EC 1.10.3.2, p-diphenolic oxidases) are extracellular enzymes belonging to the oxidoreductase group, first discovered by Yoshida (1883). They are produced in nature by many different organisms, such as fungi (Baldrian 2006), plants (Harvey and Walker 1999), bacteria (Claus and Filip 1997), and even insects (Dittmer et al 2004). Aqueous two-phase extraction (ATPE) is a well-established method of biomolecule concentration and purification. It allows for separation of biological products without the risk of denaturation, due to high water content in both phases and lack of non-polar solvents (Diamond 1990). A few review papers describing aqueous two-phase extraction properties and advances in details have been published (Raja et al 2011; Yang et al 2013; Iqbal et al 2016)
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