engleski

Evaluation of microfluidic approaches for aqueous two-phase extraction of laccase from Trametes versicolor culture filtrate

Microfluidic devices were recently found as promising tools for extractions using aqueous two-phase systems (ATPS), offering continuous process operation, precisely controlled conditions, and reduction of time and costs for obtaining necessary extraction parameters [1-3]. Aqueous two-phase systems are usually formed by mixing a polymer (usually polyethylene glycol (PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers (e.g. PEG and dextrane), and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins [4]. Several methods for purification of laccase, have been reported that consist mainly of ultrafiltration, dialysis, chromatography and electrophoresis. However, multi-step process approach results in low yield and high operating cost [5]. Similarly as in two-liquid phase biotransformations within microfluidic devices [6], liquid-liquid extractions in miniaturized systems revealed higher yields together with reduced consumption of resources and solvents as compared to macroscale systems, primarily due to smaller dimensions resulting also in reduced distances for transport to the interface and a very large surface-to-volume ratio [2, 3]. In this study a microfluidic based aqueous two-phase system (ATPS) for the extraction of laccase from the real sample is proposed. First the potential of aqueous-two phase systems (ATPS) for laccase extraction was evaluated. For this purpose, different types of ATPSs with different polymer molecular weight, salt type and pH were prepared and the partitioning coefficient of target protein laccase was preliminary tested. The obtained optimal condition were than employed, in a developed continuously operated process, within micro and meso fluidic devices for isolation and purification of the laccase from the submerged culture of Trametes versicolor Tv6 filtrate. For this purpose, rectangular Ψ-branched microchannel for parallel flow with two interfaces as well as X-junction chip for droplet formation coupled with a homemade microsettler was preliminary tested and compared. Furthermore scale-out of the droplet based system with the reduction of the equipment cost have been performed. Additionally integration of two primary separation processes namely ATPS extraction and ultrafiltration for isolating laccasse from real sample filtrate was developed (Fig. 1). Figure 1: Scheme of the continuously operating integrated ATPS extraction with ultrafiltration for isolating and concentrating of laccasse from Trametes versicolor Tv6 submerged culture filtrate.

microreactor; separation; crude laccase; ATPS

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