ObjectiveThis 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.ResultsThe 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).ConclusionsApplication of MPEC for the submerged cultures of two laccase-producing basidiomycetes proved successful in increasing of enzyme production.

The topic of integrating enzymatic reactions with in situ product removal is addressed. Different integrated reactive separations structured accordingly to the corresponding unit operations, i.e., reactive distillation, reactive chromatography, reactive crystallization, and extractive biocatalysis, are discussed. Special attention is given to their realization with homogenous and heterogeneous biocatalysis. Various enzyme immobilization techniques are presented and distinct strategies for installation of heterogeneous catalysts into process equipment are discussed.

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.

Foam fractionation process for concentration of laccases from two Basidiomycete strains under different process conditions was investigated. Culture supernatants of Cerrena unicolor and Pleurotus sapidus containing active laccase were used with and without surfactant additives. Two surfactants: cationic cetrimonium bromide (CTAB) and non-ionic Polysorbate 80 were applied in the range from 0.2 mM to 1.5 mM. The pH levels ranging from 3 to 10 were examined with particular attention to pH=4, which is close to the pI of the enzymes. Results show that the source of the enzyme is significant in terms of partitioning efficiency in a foam fractionation process. Laccase from Cerrena unicolor showed the best activity partitioning coefficients between foamate and retentate of almost 200 with yields reaching 50% for pH 7.5 and concentration of CTAB c CTAB = 0.5 mM, whereas laccase from Pleurotus sapidus showed partitioning coefficients of up to 8 with 25% yield for pH 4 and c CTAB = 0.5 mM.

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