Comparison of downstream processing methods in purification of highly active laccase.

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Continuous laccase concentration in an aqueous two-phase system.

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.

Morphology engineering of basidiomycetes for improved laccase biosynthesis.

OBJECTIVE: This work is the first application of a morphological engineering technique called microparticle-enhanced cultivation (MPEC) aimed at the facilitation of laccase production in the submerged cultures by two basidiomycetes species Cerrena unicolor and Pleurotus sapidus. RESULTS: The positive effect of the applied 10 µm Al2O3 microparticles at concentrations from 5 to 30 g Al2O3 l(-1) was shown. Laccase activity increased 3.5-fold for C. unicolor and 2-fold for P. sapidus at 15 g Al2O3 l(-1) on 9 and 14 day of the cultivation, respectively, compared to the control culture without microparticles. The increase of laccase activity in the cultivation broths was caused by the action of Al2O3 microparticles on the agglomeration of hyphae. It led to the decrease of the size of the pellets, (on average by 2 mm for C. unicolor), the change of their shape (star-shaped pellets for C. unicolor) and the change of their structure (more compact pellets for P. sapidus). CONCLUSIONS: Application of MPEC for the submerged cultures of two laccase-producing basidiomycetes proved successful in increasing of enzyme production.