The industrial and environmental applications of laccase, especially in wastewater treatment, have gained focus in recent years. Therefore, developing the proper laccase immobilization techniques, which could improve the stability of the enzymes and simplify the required downstream processes, is needed. A novel two-step immobilization process was developed, resulting in cross-linked enzyme aggregates (CLEA) in the pores of the membrane. Laccase adsorption on a biodegradable cellulose acetate microfiltration membrane along with cross-linking was investigated to maximize the enzyme load and immobilization efficiency. The optimization was done regarding the: pH, temperature, enzyme concentration, adsorption time, cross-linker concentration, and temperature. It was concluded that the highest immobilization efficiency (76%) could be achieved in acidic buffers at 29 °C with high surface activity (1174 U·m−2) at the cost of partial denaturation and membrane fouling. The membrane was successfully utilized for the enzymatic treatment of diclofenac, and 58% removal efficiency was achieved. The results indicated that cellulose acetate is a suitable carrier for adsorption-based immobilization of laccase for the potential for environmental utilisation.

Micropollutants are persistent and hazardous materials in low concentrations (ng L −1 –μg L −1 ), including substances such as pharmaceuticals, personal care products and industrial chemicals. The advancement of analytical chemistry has allowed for the detection of micropollutants; however, an efficient and economical treatment solution is yet to be installed. Fungal laccase has been a successful biocatalyst of these compounds. However, large-scale application of free enzyme is currently not feasible for removing water-borne micropollutants, partly due to relatively rapid loss in enzyme stability. In this paper, three types of cyclodextrin, α, β and γCD, were chosen to immobilise the laccase under various conditions with the aim to improve the stability of the enzyme. Laccase activity was chosen as a response parameter, and laccase-cyclodextrin binding was evaluated by Fourier-transform infrared spectroscopy (FTIR). Results showed an optimum using α-cyclodextrin immobilisation. At that level, α-cyclodextrin increased the half-life of laccase and slightly improved its activity in all tested pH by physically bonding to laccase. By protecting the enzyme structure, activity was maintained under a range of circumstances (acidic conditions, from 10 to 50 °C). Under room temperature and at pH 5, α-cyclodextrin-laccase nanocomposite had a better removal efficiency of diclofenac compared to free laccase of the same concentration. Graphical abstract

Multiwalled carbon nanotubes (MWCNTs) were oxidized using a mixture of H2SO4 and HNO3, and the oxidized MWCNTS were decorated with magnetite (Fe3O4). Finally, poly -N-isopropyl acrylamide-co-butyl acrylate (PNIPAM) was added to obtain P-NIPAM/Fe/MWCNT nanocomposites. The nanosorbents were characterized by various techniques, including X-ray diffraction, transmission electron microscopy, scanning electron microscopy, thermogravimetric analysis, and Brunauer-Emmett-Teller analysis. The P-NIPAM/Fe/MWCNT nanocomposites exhibited increased surface hydrophobicity. Owing to their higher adsorption capacity, their kerosene removal efficiency was 95%; by contrast, the as-prepared, oxidized, and magnetite-decorated MWCNTs had removal efficiencies of 45%, 55%, and 68%, respectively. The P-NIPAM/Fe/MWCNT nanocomposites exhibited a sorbent capacity of 8.1 g/g for kerosene removal from water. The highest kerosene removal efficiency from water was obtained at a process time of 45 min, sorbent dose of 0.005 g, solution temperature of 40 ?, and pH 3.5. The P-NIPAM/Fe/MWCNTs showed excellent stability after four cycles of kerosene removal from water followed by regeneration. The reason may be the increase in the positive charge of the polymer at pH 3.5 and the increased adsorption affinity of the adsorbent toward the kerosene contaminant. The pseudo second-order model was found to be the most suitable model for studying the kinetics of the adsorption reaction.

Organic micropollutants like pharmaceuticals, personal care products, pesticides and industrial chemicals have received an ever growing attention due to their adverse ecological effects in natural water bodies. A promising technology to remove the micropollutants from the wastewater is biocatalytic treatment using oxidoreductase enzymes. In order to improve removal efficiency and feasibility, the focus has been on developing enzyme-mediator systems and immobilisation techniques. Nonetheless, since oxidoreductase enzymes often promote coupling reactions, enzymatic reactions do not necessarily result in simpler compounds. Thus transformation products, which might be in equally or even more hazardous pollutants, can be still present in the wastewater after treatment. Therefore, the toxicity and ecological effects of the reaction products have to be investigated rigorously to ensure that the treatment mitigates the environmental and health related impacts.

The elimination or degradation of micropollutants from wastewater is becoming ever more important nowadays. Using oxidoreductase enzymes to treat different micropollutants seems a promising solution. However, the viability of the process is highly dependent on the availability and stability of the applied enzymes. In order to improve the stability and provide faster reaction rates, enzymes can be immobilized in various carriers. Properties such as simple production, easy retention and biodegradable carrier material are advantageous, e.g. entrapping laccase in alginate beads. This paper shows the results of the preparation and characterization of immobilized laccase entrapped in calcium alginate beads. The technique of adding a mixture of sodium alginate and laccase dropwise into calcium chloride has been applied, improved and standardized to produce laccase-containing beads of uniform size and activity. For the purpose of characterization, a widely used substrate, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, was used to evaluate the performance of the laccase-containing alginate beads. In addition to the characterization of the laccase-containing alginate beads, the enzyme kinetic constants (= 26.43 µM, ax = 0.23 µM/min) were determined. The reduction in the activity during storage has been described by a decay constant (0.26 d−1 ) that provides information concerning the design constraints of the process. Results will be used to test the method in terms of the removal of organic micropollutants in continuous systems.