Rational Design and Directed Evolution

Rational methods are based on experimental evidence that the effect of single amino acid substitutions on protein stability can be well approximated as additive, distributed, and large independent interactions. Moreover, by comparison of homologous enzymes from thermophilic and nonthermophilic microorganisms, it is known that introduction of disulfide bridges as well as increased numbers of proline residues increase protein stability. 

Site-directed mutagenesis was used to substitute the His residue in position 775 to Asn (His775Asn). This mutation improves the substrate specificity but simultaneously causes a decrease in thermal stability. Some regions of A. calcoaceticus that are responsible for EDTA resistance and for thermal stability were inserted into the E. coli gene by homologous recombination. 

The chimeric genes obtained were used to transform E. coli cells, with each single clone expressing one variant of PQQ-glucose dehydrogenase. The enzyme was isolated and assayed in a buffer containing PQQ to form the holoenzyme, and a colorimetric indicator. Enzymatic activity was assayed for different substrates, and the effect of EDTA and thermal stability were investigated. When 

From bacterial cultures, cut and amplified PQQGDH genes 

Change single codon ( ) and homologous recombination (rational design, knowledge supported) 

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Lehmann M, Wyss M. Engineering proteins for thermostability the use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 2001 12 371-375. Berezovsky IN, Shakhnovich El. Physics and evolution of thermophilic adaptation. Proc. Natl. Acad. Sci. U.S.A. 2005 102 12742-12747. 

Fig. 1 Comparison between rational design and directed evolution. (View this art in color at www.dekker.com.)...

Hybrid Approaches Combining Rational Design and Directed Evolution... 

Fig. 6 Schematic representation of hybrid approaches combining rational design and directed evolution. Rational design may allow a large jump in sequence space from high fitness for function A to a low fitness for novel function B. The low fitness for function B might be readily optimized by directed evolution.

Directed evolution proved to be a rapid yet powerful method to alter enzyme properties or to develop enzymes with novel properties, without requiring knowledge of the enzyme structure and catalytic mechanism. Arnold (2001) has an impressive review on enzyme performance improvement by a combination of rational design and directed evolution. Directed evolution applied to lipases has been reviewed by different authors (Petrounia and Arnold 2000 Tobin et al. 2000 Jaeger et al. 2001). Directed evolution has been employed for the creation of... 

Enzyme engineering is undergoing the most profound and exciting transformation in its history and promises unprecedented expansion in the scope and applications of modified or improved enzymes with desired physical and catalytic properties. Two complementary strategies are currently available rational design and directed evolution. Although both approaches have been met with great success, each has its limitations (13). 

Protein Engineering. The field of protein engineering involves developing and creating useful or important proteins. Protein engineering encompasses two main strategies rational design and directed evolution. 

Basic research will further develop techniques for studies on electron transfer phenomena in biological macromolecules. In applied research, the optimization of the direct electron transfer between bioelements and electrodes is dominant. Improvement of DET is anticipated by a better orientation and binding of redox proteins and redox enzymes and the stabilization of the bioelement. Simultaneously an increased stability is pursued. The employed genetic methods for protein engineering are based oti rational design and directed evolution. With the advance of analytical methods and a better knowledge of the structure/functional relationship of DET in redox proteins and redox enzymes the engineering of electron transfer pathways within proteins will become feasible. 

In nature, enzymes play an important role in the survival and reproduction of their source organism. Therefore, many enzymes in their natural form are not suitable for application directly as biocatalysts in bioprocessing. For example, most enzymes are active at relatively mild conditions, thus may not be viable under the harsher conditions encountered in most indnstrial production systems. Various methods have been used to improve the properties of enzymes, including selectivity, activity and thermostability, in order to enable them to function as efficient industrial biocatalysts. Enzyme engineering, which encompasses rational design and directed evolution, is an efficient method to improve enzyme properties. Rational design seeks for beneficial mutations or protein sequences by applying empirically derived rules or theoretical models. Meanwhile, directed evolution uses a combinatorial approach to create libraries of enzymes from which enhanced variants can be identified... 

HNLs often exhibit only low activity toward industrially relevant compounds. Thus, a lot of effort was (and still is) made to engineer HNLs for the acceptance of unfavorable substrates by rational design and directed evolution. 

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