Figure 1. Example for the identification of an enzyme-encoding gene. First, degenerate primers are designed based on an alignment of homologous proteins (using BLAST and ClustalW, see sections below). These primers are then used to amplify a sequence fragment from genomic DNA of the microorganism. In order to get access to the full-length gene, various methods such as gene walking, homologous PCR, inverse PCR, or site-finding PCR can be used. Once the entire gene has been identified, it can be subcloned, transformed, and expressed in the recombinant host organism.

Figure 2. Overview of approaches for protein discovery and engineering by rational, evolutionary, or combined methods (taken from Behrens et al., 2011).

Figure 3. Inversion of the stereospecificity of N-acetylneuraminic acid aldolase (NeuAc) toward sialic acid by directed evolution (Wada et al., 2003).

Figure 4. Starting from a given protein sequence, the software tool BLAST enables identification of related proteins (top) as exemplified here for esterases. These can then be compared in detail (bottom) using alignment software such as ClustalW to identify identical (*), homologous (. or:), and differing amino acids (blank) as well as conserved motifs.

Figure 5. Principle of directed evolution. The gene(s) encoding the wild-type or homologous enzyme(s) are subjected to random mutagenesis using nonrecombining or recombining methods. The resulting mutant libraries are then cloned and expressed (often in microtiter plates). The desired improved variants are identified by high-throughput screening systems, usually using microtiter plate-based assays or selection (e.g., using agar plate assays; not shown).

Figure 6. Homology between mutants of a cephalosporinase gene obtained by DNA shuffling of parental genes from four different species (Crameri et al., 1998).

Figure 7. Fluorogenic assay based on umbelliferone derivatives. Enzyme activity yields a product, which upon oxidation with sodium periodate and treatment with bovine serum albumin (BSA) yields umbelliferone (Reymond and Wahler, 2002).

Figure 8. Overview of the directed evolution of a lipase from P. aeruginosa for the enantioselective resolution of 2-methyl decanoate. In the first step (1), the lipase gene was subjected to random mutagenesis; next, the mutated genes were expressed and secreted (2). Screening for improved enantioselectivity was based on a spectrophotometric assay using optically pure (R)- or (S)-p-nitrophenyl esters of the substrate (3). Hit mutants with improved enantioselectivity were then verified by gas chromatography (4). The cycle was repeated several times to identify best mutants (5) (Reetz et al., 2001).

Figure 9. Changes in enantioselectivity of a lipase from P. aeruginosa using methods of directed evolution. Starting from the nonselective wild type (WT, E = 1.1), the combination of various genetic tools led to the creation and identification of variants with high (S)-selectivity (E = 51) and with good (R)-selectivity (E = 30) (Reetz et al., 2001).