*5.2. Enzyme Engineering*

Protein engineering can be used to improve the catalytic performance of enzymes [169,170]. Rational design, directed evolution and combinations of these approaches are widely used to prepare more active and stable enzymes [170]. In rational design, mutations are inserted into specific locations in the protein through site-directed mutagenesis. The lack of a full understanding of the relationship between the protein structure and the rate of electron transfer makes it difficult to improve the catalytic activity using rational design. The structural knowledge of proteins is not required for directed evolution and catalytic activity can be improved in the absence of a detailed crystal structure of the enzyme by mimicking Darwinian evolution [170]. Modifying the heme centre of myoglobin increased its dehalogenation activity to over 1000 times that of a native dehaloperoxidase [171]. Recently, Chen et al. prepared cytochrome P450 enzymes through the directed evolution of serine-ligated P450 variants for the preparation of cyclopropene with high efficiency (with a total turnover number (TTN) of up to 5760) and high selectivity (>99.9% ee) [172]. Brandenberg et al. reported a cytochrome P450 variant that was able to catalyse the C2-amidation of indole. Both the heme and reductase domains were modified, improving the catalytic activity with the total turnover number increasing from 100 to 8400 and the product yield from 2.1 to 90% [173]. Mateljak et al. used computational design with directed evolution to design fungal high-redox-potential laccases that exhibited high stability and activity toward redox mediators [174]. Further work was performed to investigate the use of these laccases for the reduction of O<sup>2</sup> in the presence and absence of ABTS. The designed laccases could reduce O<sup>2</sup> at low overpotentials [175]. Protein engineering has been used to adjust the properties of NAD(P)H-dependent oxidoreductases [176]. For example, Liu et al. used directed evolution to prepare an NADH-dependent alcohol dehydrogenase from *Lactococcus lactis* for the production of isobutanol. The catalytic efficiency of the engineered enzyme increased by a factor of 160 in comparison with the wild-type enzyme [177]. Li et al. used directed evolution to improve the catalytic activity of puritative oxidreductase in the production of 1,3-propanediol [178].
