Protein engineering rational enzyme design

Another method used in protein engineering is directed evolution. Unlike rational enzyme design, this shategy requires neither structural information nor the knowledge of basic enzyme funchons. But there is a need for screening or selection... 

Transaminases are most powerful tools for the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter several approaches for tiie preparation of fine chemicals or building blocks for pharmaceuticals were discussed, like asymmetric synthesis or kinetic resolution. The main limitations of transaminase-catalyzed reactions are the need to shift the equihbrium to the product side and substrate and product inhibition. Some solutions to overcome such inhibition were presented here for example, multienzyme cascades or biphasic extraction of the product. Protein engineering by directed evolution or rational enzyme design is a promising option to find transaminases with different substrate specificities and enantiopreferences. This is becoming more and more important for the pharmaceutical industry. Furthermore, it is a way to alter enzyme properties known so far, like thermostability and solvent and pH stability. Protein engineering has been assisted by the recently solved structures of certain transaminases. 

The last systematic description of heme peroxidases was published in 1999 by Brian Dunford, from the University of Alberta in Canada. The book Heme peroxidases covers discussion on three-dimensional structure, reaction mechanism, kinetics, and spectral properties of representative enzymes from bacterial, plant, fungal, and animal origin. Since 1999, vast information on basic but also applied aspects of heme peroxidases has been generated. We believe fusion of these two aspects will benefit research of those dedicated to development of biocatalytic process. The aim of this book is to present recent advances on basic aspects such as evolution, structure-function relation, and catalytic mechanism, as well as applied aspects, such as bioreactor and protein engineering, in order to provide the tools for rational design of enhanced biocatalysts and biocatalytic processes. The book does not include an exhaustive listing of references but rather a selected collection to enrich discussion and to allow envisioning future directions for research. 

Fig. 13.3. Correlation of required mechanistic information, required structural information, importance of screening and number of possible enzyme variants in protein engineering by rational protein design and directed evolution.

In the present chapter, we will focus on the more rational approaches of enzyme engineering and design. Basic techniques for site-directed mutagenesis, protein crystallization, and comparative modeling will also be introduced. Some recent, key examples of rational protein engineering will be described in a somewhat detailed manner. There are also very informative reviews in the literature[11, 15 17]. 

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. 

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