Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

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The ability of proteins and peptides to undergo proteolysis is essential to their biological function. Herein, we report the first detailed study of the protease reactivity of peptide dendrimers. Dendrimers are regularly ramified, tree-like synthetic macromolecules with promising application in technology and medicine. Using trypsin and alpha-chymotrypsin cleavage sites as models, we show that the protease reactivity of peptide dendrimers can be controlled by the degree of branching. Dendrimers with two or three amino acids between branching points were readily cleaved by trypsin irrespective of the position of the reactive sequence within the dendrimers, for example in D1, (Ac-Gly-Phe-Pro)4(Dap-Hyp-Arg[downward arrow]Met)2Dap-Ser-Gly-betaAla-NH2, and D12, (Ac-Ser-Ala)8(Dap-Ala-Arg[downward arrow])4(Dap-Ala-Asp)2Dap-Phe-Ala-Lys*-NH2 (Dap: (S)-2,3-diaminopropionic acid branching point, Hyp: hydroxyproline, Lys*: FITC-labeled lysine, [downward arrow]: cleavage site). On the other hand cleavage was blocked in more compact dendrimers with only one amino acid between branching points, for example in D18B, (Ac-Glu)8(Dap-Phe)4(Dap-Arg)2Dap-Leu-NH2). The control of proteolysis by topology provides a novel possibility to tune the biological properties of peptide dendrimers not available in linear peptides, and should be generally useful for their use as functional biomolecule analogues, for example, in the context of drug delivery applications.

A combinatorial library of 15,536 cyclic decapeptide analogues of tyrocidine A and gramicidin S was prepared on photocleavable TentaGel beads. The beads were photolyzed without solvent, and spread onto an agar plate inoculated with bacterial lawn. Clear zones were formed around beads carrying antimicrobially active peptides such as E18 c(kVOrnLfThiYOrnLq), which inhibited growth of B. subtilis and selected MRSA strains.

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