Protein engineering enzyme selection

Enzymes. Protein engineering has been used both to understand enzyme mechanism and to selectively modify enzyme function (4,5,62—67). Much as in protein stabiUty studies, the role of a particular amino acid can be assessed by replacement of a residue incapable of performing the same function. An understanding of how the enzyme catalyzes a given reaction provides the basis for manipulating the activity or specificity. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

The term medium engineering , that is the possibility to affect enzyme selectivity simply by changing the solvent in which the reaction is carried out, was coined by Klibanov, who indicated it as an alternative or an integration to protein engineering [5aj. Indeed, several authors have confirmed that the enantio-, prochiral-, and even regioselectivity of enzymes can be influenced, sometimes very remarkably, by the nature of the organic solvent used. 

The experimental evidences that medium engineering might represent an efficient method to modify or improve enzyme selectivity (alternative to protein engineering and to the time-consuming search for new catalysts) were immediately matched by the search for a sound rationale of this phenomenon. The different hypotheses formulated to try to rationalize the effects of the solvent on enzymatic enantioselectivity can be grouped into three different classes. The first hypothesis suggests that... 

The design of supramolecular catalysts may make use of biological materials and processes for tailoring appropriate recognition sites and achieving high rates and selectivities of reactions. Modified enzymes obtained by chemical mutation [5.70] or by protein engineering [5.71] represent biochemical approaches to artificial catalysts. 

The by-products, such as isomaltose, have to be removed chromatographically, which is energy- and cost-intensive. Protein engineering focuses on increasing the glucose yield (affecting enzyme selectivity) and enzyme thermostability. 

A breakthrough in recombinant DNA technology and protein engineering was achieved by recognizing that the process of natural selection can be harnessed to evolve effective enzymes in artificial circumstances. In this framework of directed evolution , the processes of natural evolution for selecting proteins with the desired properties are accelerated in a test tube. The starting point is an enzyme with a measurable desired activity which still has to be improved. 

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. 

For example, see Y. Wei and M. H. Hecht. Enzyme-like proteins from an unselected library of designed amino acid sequences. Protein Engineering Design and Selection, 17 (2004), 67-75. 

The use of a-transglucosidases in the large-scale manufacture of novel bioderivatives is stiU limited by several factors such as enzyme selectivity, stability, and, in some cases, efficiency. To overcome these Umitations and further enlarge the appUcatimis of these enzymes, the latest protein engineering technologies have been used to tailor biocatalysts with specific properties for novel oligosaccharide. 

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