Refolding of laccase from Trametes versicolor using aqueous two phase systems: Effect of different additives

Protein refolding is a strategy used to obtain active forms of proteins from inclusion bodies. On its part, laccase is an enzyme with potential for different biotechnological applications but there are few reports regarding its refolding which in many cases is considered inefficient due to the poor obtained refolding yields. Aqueous Two-Phase Systems (ATPS) have been used for the refolding of proteins getting acceptable recovery percentages since PEG presents capacity to avoid protein aggregation. In this work, 48 PEG-phosphate ATPS were analyzed to study the impact of different parameters (i.e. tie line length (TLL), volume ratio (V_R) and PEG molecular weight) upon the recovery and refolding of laccase. Additionally, since laccase is a metalloprotein, the use of additives (individually and in mixture) was studied with the aim of favoring refolding. Results showed that laccase presents a high affinity for the PEG-rich phase obtaining recovery values of up to 90%. Such affinity increases with increasing TLL and decreases when PEG molecular weight and V_R increase. In denatured state, this PEG-rich phase affinity decreases drastically. However, the use of additives such as l-cysteine, glutathione oxidized, cysteamine and Cu⁺² was critical in improving refolding yield values up to 100%. The best conditions for the refolding of laccase were obtained using the PEG 400gmol^-1, TLL 45% w/w, V_R 3 ATPS and a mixture of 2.5mM cysteamine with 1mM Cu⁺². To our knowledge, this is the first time that the use of additives and the behavior of the mixture of such additives to enhance refolding performance in ATPS is reported.