Enhanced Laccase Separation from Fermentation Medium Using Cryogel Columns

The laccase enzyme family, a member of the oxidoreductase enzyme class, is one of the most commercially valuable enzymes and responsible for the catalysis of the oxidation of single electrons of a wide variety of phenolic molecules (Kallio et al., 2009). Laccases are eco-friendly since they require molecular oxygen as co-substrate in order to catalyze wide range of reactions and yield water as the sole by-product (Chandra and Chowdhary, 2015, Mayolo‐Deloisa et al., 2015, Yavaşer and Karagözler, 2021).

Laccases can be found in a wide variety of sources including bacteria, fungi, and plants. Extracellular laccases are commonly purified from various fungal sources (Ko et al., 2001, Thurston, 1994). Aspergillus niger (A. niger) species are reported to produce laccase and revealed to be a good source of laccases (Ferraroni et al., 2017, Gianfreda et al., 1999). Laccases are utilized in food processing, biosensor and analytical applications, textile color conversion, dye decolorization, pulp delignification, pesticide or insecticide degradation, organic synthesis, bioremediation, biodegradation of environmental phenolic pollutants and waste detoxification, due to their versatility in acting on different substrates (Couto and Herrera, 2006). Since the textile, pulp, and paper industries generate a huge amount of waste, removing these wastes has become a major issue. Laccase was discovered as an enzyme that detoxifies waste while being environmentally friendly. In the food industry, laccases have been used for the elimination of unwanted phenolic compounds in cooking, juice processing, wine stabilization, and wastewater bioremediation (Couto and Herrera, 2006, Gianfreda et al., 1999). In the brewing industry, laccase provides stability and increases the shelf life of beer. Laccase is widely used in fruit juice stabilization, and the naturally occurring phenol compounds and their oxidative products in fruit juice give color and flavor to the juice (Madhavi and Lele, 2009). Laccase also can reduce odor from landfills, livestock farms and pulp mills. Laccase, on the other hand, can catalyze electron transfer reactions without the need for additional cofactors and has some distinct advantages over other enzymes, such as the ability to oxidize phenolic compounds in the presence of oxygen (Rodríguez-Delgado et al., 2015). Since laccases catalyze electron transfer reactions without additional cofactors, they can also be used as biosensors to detect various phenolic compounds, oxygen and azide (Kunamneni et al., 2007).

A wide variety of techniques have been employed for the purification of laccases for many years, including ammonium sulfate-based protein precipitation, anion exchange chromatography and gel filtration chromatography. Among these, the most widely used technique for laccase purification is salt elution from an anion-exchange resin (Patel et al., 2014, Yuan et al., 2016). Production and purification of laccases are laborious and time-consuming processes, and often require high costs, and their low efficiency recovery for further use have been a challenging issue (Patel et al., 2014). Thus, alternative materials and techniques are still required to overcome these drawbacks.

Cryogels are support materials synthesized at sub-zero temperatures, featured with characteristics such as interlinked macro- and micropores, excellent swelling behavior and reusability due to their chemical and mechanical stability. Interlinked macroporous structure of cryogels provides ease of diffusion and mass transfer (Andaç et al., 2016, Zenger and Peşint, 2022). Large pore sizes make cryogels advantageous materials in biotechnology and medical fields for many different purposes such as decomposer, carrier and bioreactor (Dainiak et al., 2009). Hereby, cryogels have been extensively used as chromatographic absorbent, support material, artificial cell tissues and drug delivery systems (Dainiak et al., 2009, Gun’ko et al., 2013, Kuyukina et al., 2009, Mattiasson et al., 2009). Water-based macroporous cryogels exhibit an excellent biocompatibility and they can be produced in different size and shape, so they are frequently preferred materials in a wide range of applications (Mishra et al., 2016, Sharma et al., 2013). There are many studies reported in literature on various enzyme purification such as, carbonic anhydrase, lysozyme, horse radish peroxidase etc., using cryogels (Çimen et al., 2020, Keçili et al., 2018, Peşint et al., 2021, Uygun et al., 2014), however laccase purification studies were not reported using cryogels, to the best of our knowledge.

In this study, 1-vinylimidazole (VIM) functionalized poly (2-hydroxyethyl methacrylate) (PHEMA) based cryogel column (PHEMA-VIM) was synthesized for the purification of laccase produced by A. niger from crude medium.

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