Hydrogels with 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), 17α-ethinylestradiol (EED), triclosan, paracetamol, and 4-tert-octylphenol]. 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.
In this study, solvogels containing (2-((2-(ethoxycarbonyl)prop-2-en-1-yl)oxy)-ethyl) phosphonic acid (ECPA) and N,N′-diethyl-1,3-bis-(acrylamido)propane (BNEAA) as the crosslinker are synthesized by UV induced crosslinking photopolymerization in various solvents. The polymerization of the ECPA monomer is monitored by the conversion of double bonds with in situ attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The morphology of the networks is characterized by in situ photorheology, solid state NMR spectroscopy, and scanning electron microscopy (SEM) of the dried gels. It is demonstrated that the storage modulus is not only determined by the crosslinker content in the gel, but also by the solvent used for preparation. The networks turn out to be porous structures with G′ being governed by a rigid, phase-separated polymer phase rather than by entropic elasticity. The external and internal pKa values of the poly(ECPA-co-BNEAA) gels were determined by titration with a specially designed method and compared to the calculated values. The polymer-immobilized phosphonic acid groups in the hydrogels induce buffering behavior into the system without using a dissolved buffer. The calcium accumulation in the gels is studied by means of a double diffusion cell filled with calcium ion-containing solutions. The successful accumulation of hydroxyapatite within the gels is shown by a combination of SEM, energy-dispersive X-ray spectroscopy (EDX) and wide-angle X-ray scattering (WAXS).
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