Urease-induced calcification of segmented polymer hydrogels - a step towards artificial biomineralization.

Natural organic/inorganic composites, such as nacre, bones and teeth, are perfectly designed materials with exceptional mechanical properties. Numerous approaches have been taken to synthetically prepare such composites. The presented work describes a new way of mineralizing bulk materials on a large scale following the approach of bioinduced mineralization. To this end, a series of polymer conetworks with entrapped urease were prepared. After polymerization, the entrapped urease shows high enzymatic activity. The bioactive polymer conetworks were then treated with an aqueous mixture of urea and CaCl2. The urease-induced calcification indeed allows formation of carbonate crystals exclusively within the hydrogel even at room temperature. The influence of network composition, degree of cross-linking, immobilized urease concentration and temperature of calcification were investigated. By varying these parameters, spherical, monolithic clusters, as well as bar-like nanocrystals with different aspect ratios in spherical or dendritic arrays, are formed. The grown nanocrystals improve the stiffness of the starting material by up to 700-fold, provided that the microstructure shows a dense construction without pores and strong interaction between crystals and network. The process has the potential to generate a new class of hybrid materials that would be available on the macroscopic scale for use in lightweight design and medicine.

Investigations on diffusion limitations of biocatalyzed reactions in amphiphilic polymer conetworks in organic solvents.

The use of enzymes as biocatalysts in organic media is an important issue in modern white biotechnology. However, their low activity and stability in those media often limits their full-scale application. Amphiphilic polymer conetworks (APCNs) have been shown to greatly activate entrapped enzymes in organic solvents. Since these nanostructured materials are not porous, the bioactivity of the conetworks is strongly limited by diffusion of substrate and product. The present manuscript describes two different APCNs as nanostructured microparticles, which showed greatly increased activities of entrapped enzymes compared to those of the already activating membranes and larger particles. We demonstrated this on the example of APCN particles based on PHEA-l-PDMS loaded with α-Chymotrypsin, which resulted in an up to 28,000-fold higher activity of the enzyme compared to the enzyme powder. Furthermore, lipase from Rhizomucor miehei entrapped in particles based on PHEA-l-PEtOx was tested in n-heptane, chloroform, and substrate. Specific activities in smaller particles were 10- to 100-fold higher in comparison to the native enzyme. The carrier activity of PHEA-l-PEtOx microparticles was tenfold higher with some 25-50-fold lower enzyme content compared to a commercial product.

Solid-solid interface adsorption of proteins and enzymes in nanophase-separated amphiphilic conetworks.

Amphiphilic polymer conetworks (APCNs) are materials with a very large interface between their hydrophilic and hydrophobic phases due to their nanophase-separated morphologies. Proteins were found to enrich in APCNs by up to 2 orders of magnitude when incubated in aqueous protein solutions, raising the question of the driving force of protein uptake into APCNs. The loading of poly(2-hydroxyethyl acrylate)-linked by-poly(dimethylsiloxane) (PHEA-l-PDMS) with heme proteins (myoglobin, horseradish peroxidase, hemoglobin) and lipases was studied under variation of parameters such as incubation time, pH, concentration of the protein solution, and conetwork composition. Adsorption of enzymes to the uncharged interface is the main reason for protein uptake, resulting in protein loading of up to 23 wt %. Experimental results were supported by computation of electrostatic potential maps of a lipase, indicating that hydrophobic patches are responsible for the adsorption to the interface. The findings underscore the potential of enzyme-loaded APCNs in biocatalysis and as sensors.