In the past ™molecular imprinting∫ was mainly confined to the separation of organic molecules and to nonenzymatic catalysis.[1±3] With advancements in this technology, its potential and utility in the field of biotechnology was also realized. [4] In order to improve its applicability, new variations in the conventional imprinting technique have emerged, such as ™bioimprinting∫, [5, 6] metal chelation imprinting, [7] affinity imprinting, [8,9] and a combination of immobilization and ™bioimprinting∫. [10,11] The memory effect of enzymes caused by bioimprinting without a further immobilization step was found to depend on the water content of the medium and was completely lost when the reaction was carried out in the presence of a certain amount of water. [5, 12,13] This was exemplified by the fact that, in aqueous medium, renaturation of a bioimprinted protein to its original native conformation resulted in the loss of imprinted memory. [5,13] However, water is an indispensable milieu for most enzymatic reactions. Therefore, our research is focused on how to maintain the ™bioimprinted memory∫ of an enzyme not only in organic solvents but also in aqueous solution systems. Here we demonstrate that the combinatorial crosslinked imprinting approach (we termed it CLIP) overcomes this problem. The CLIP methodology is shown schematically in Scheme 1. Keyes et al. employed a different kind of methodology to alter the catalytic properties of enzymes. [14] In their strategy, the enzyme complexed with ligands was crosslinked by using glutaraldehyde. This whole process was carried out in aqueous buffer, probably because the present imprinting concept in organic solvents was not known to that time.In our initial attempts [10] monomeric, low-molecular-weight (26 ± 28 kDa), nonglycosylated and cofactor-independent proteolytic enzymes, such as chymotrypsin and subtilisin, were selected to demonstrate the feasibility of the CLIP approach to rationally modify their catalytic properties. These proteases, when ™bioimprinted∫ with N-acetyl-D-tryptophan, can accept both D-and L-configured substrates, whereas the native enzyme only recognizes the L-form for synthesis of its ethyl ester in dry Scheme 1. Schematic illustration of combinatorial crosslinked imprinting methodology (CLIP). The enzyme of interest is first derivatized and then complexed by using ligands such as substrate analogues or inhibitors in aqueous medium. In the next step, imprinted memory is created by precipitation of protein and drying under vacuum. Subsequently, this imprinted memory is covalently ™frozen∫ by crosslinking the precipitated protein in dry organic solvent. The resulting CLIP enzyme is washed to remove the ligand. It can then be used either in aqueous medium or organic solvent. In the present case of glucose oxidase (GO), the ligand was its competitive inhibitor D-galactose, and the novel catalytic property in aqueous medium was acceptance of D-galactose as a substrate to give D-galactono-1,4-lactone as a product.cyclohexane. [5,10] Hydrolysis of the...
Ultrasonic resonator technology (URT) was compared with the well established UV-Vis/ninhydrin assay to estimate protease activities in defined buffer systems. Hydrolysis of casein was measured using subtilisin, trypsin, halophilic protease from Haloferax mediterranei and Bacillus lentus alkaline protease. Sensitivity, reproducibility, working range as well as the limit of detection and the limit of quantification were comparable for both methods. Salt concentrations (0.5 M NaCl) interfered with the URT method. The quantification of protease activity by URT was possible when the product concentration measured by the UV-Vis/ninhydrin assay was correlated to the corresponding ultrasonic velocity signals.
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