Chemical Design of Hydrogels with Immobilized Laccase for the Reduction of Persistent Trace Compounds in Wastewater

Hydrogels immobilized enzymes are increasingly applied in biocatalytic industrial processes. Here, polymer hydrogels containing 2-hydroxyethyl methacrylate (HEMA), itaconic acid (ITA), (2-((2-(ethoxycarbonyl)prop-2-en-1-yl)oxy)ethyl) phosphonic acid (ECPPA), and N,N'-diethyl-1,3-bis(acrylamido)propane (BAAP) as the cross-linker are synthesized by UV-initiated radical polymerization. Laccase from Trametes versicolor (LAC) is modified by reaction with itaconic anhydride (ITAn) yielding the LAC-immobilized monomer ITA-LAC with enhanced enzyme activity. ITA-LAC paves the way to an in situ method for enzyme immobilization. Hydrogels with HEMA, ECPPA, and BAAP with stepwise varied chemical composition and functionalization are prepared. The influence of the composition on the morphology, the swelling behavior, the mechanical stability, and the enzymatic activity is studied. The polymerization is monitored by the conversion of double bonds with in situ attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The polymerization of HEMA is complete after 10 min of UV exposure, whereas hydrogels of HEMA/ITA/ECPPA (85/5/10) with 5 mol % cross-linker require 30 min. These hydrogels are compared with those containing ITA-LAC instead of ITA. The covalent binding of LAC is proven by ATR-FTIR spectroscopy and results in an enhanced enzyme activity. The incorporation of ECPPA induces pH-dependent swelling with an equilibrium degree of swelling of up to 6 at pH 8. Only a weak influence of temperature on the degree of swelling is found. The morphology strongly depends on the hydrogel composition. LAC-ITA hydrogels are characterized by an open morphology providing access to catalytic centers. The enzyme-immobilized hydrogels are used as granules as well as coatings on porous Al2O3 ceramic substrates as biocatalysts to convert models for organic trace compounds [bisphenol A (BPA), diclofenac, p-chlorophenol (pCP), 17 alpha-ethinylestradiol (EED), triclosan, paracetamol, and 4-tertoctylphenol]. The highest conversion after 24 h in water is achieved for triclosan (>90%), while pCP, BPA, and EED reach a conversion between 60% and 70%. The conversions are even higher in citrate buffer.

Automated summary

Hydrogels immobilized with enzymes are synthesized using UV-initiated radical polymerization with HEMA, ITA, ECPPA, and BAAP. ITA-LAC modification enhances enzyme activity and pH-dependent swelling behavior.