Biocatalysis Dr Rebecca Buller https www zhaw chdelsfmforschungchemieundbiotechnologieccbiocompetencecenter

Biocatalysis, Dr. Rebecca Buller https: //www. zhaw. ch/de/lsfm/forschung/chemie-und-biotechnologie/ccbio-competence-center/ Protein Engineering Background In the early days of biotechnology, the biocatalytic reaction was adjusted to meet the optimal conditions for the enzymes used. Nowadays, we are able to engineer the enzymes themselves. Via this method, called protein engineering, the enzymes are matched to the target substrate or to the reaction condition. Furthermore, protein engineering can even introduce alternative reaction mechanisms for a given enzyme. Two complementary approaches can be applied: rational protein design and directed evolution. For rational design, a large set of enzymatic parameters is needed, but only a few hotspots in the sequence are mutated, which keeps the screening effort low. For directed evolution, however, only basic information about the protein is required, because the gene sequence is mutated randomly, but the screening effort is very high (Fig. 1). Fig. 1 Methodologies for protein engineering (adapted from S. Lutz, U. T. Bornscheuer, Protein Engineering Handbook, Vol. 3, Wiley-VCH, 2012) Gene/DNA shuffling In this method, homologues genes are recombined in vitro in order to propagate the beneficial mutations. Different members of the gene family are fragmented using DNase 1 followed by PCR. During PCR, different members of the family are cross-primed. DNA fragments with high homology will anneal to each other. The generated hybrids are then used to generate a library of mutants, which are tested for unique properties (Fig. 2). In summary: Fig. 2 Gene shuffling (J. Cohen Science Vol. 293, Issue 5528, 237, 2001) • Sequence homology is required • Several parent genes can be used • Creation of chimeras is possible • Useful mutations are combined, harmful ones lost Circular permutation An alternative way to engineer proteins is the circular permutation (Fig. 3). Here, the native protein termini will be connected via a covalent linker. Afterwards, new ends are introduced through the cleavage of an existing peptide bond. Circular permutation can perturb local tertiary structure and protein dynamics, as well as introduce possible quaternary structure changes. In several recent studies, these effects have successfully been exploited to manipulate protein scaffolds, resulting in improved catalytic activity and altered substrate or ligand binding affinity, as well as enabling the design of novel biocatalysts and biosensors. Fig. 3 Circular permutation (F. H. Arnold Nature Biotechnology Vol. 24, 328 – 330, 2006)