Enzyme protein engineering

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. 

Figure 2. Efficiency of an rDNA approach to enzyme commercialization. Areas in boxes refer to the typical times required for Identification, Scale-up and Commercialization. Efficiencies are recognized in Identification of enzymes (protein engineering) and shortened Scale-up times (generic hosts).

Change individual amino acids in the enzyme and thereby improve the physical properties or performance of the enzyme (protein engineering)... 

Carl Johnson That is certainly an important area. Aside from attempts to mimic enzymes, protein engineering is available to improve enzymes. It may surprise some in the audience to know that their shirts have been washed with detergents that contain enzymes. Many of these are not Nature s enzymes but are enzymes that have been engineered and produced in huge commercial scales. An... 

Biological catalysts — enzymes — are usually proteins. The development of new protein syntheses is nowadays dominated by genetic protein engineering (see section 4.1.2.6). Bio-organic approaches towards novel catalytically active structures and replicating systems try to manage without biopolymers. 

Many enzymes have been the subject of protein engineering studies, including several that are important in medicine and industry, eg, lysozyme, trypsin, and cytochrome P450. SubtiHsin, a bacterial serine protease used in detergents, foods, and the manufacture of leather goods, has been particularly well studied (68). This emphasis is in part owing to the wealth of stmctural and mechanistic information that is available for this enzyme. 

Specificity for a particular charged substrate can be engineered into an enzyme by replacement of residues within the enzyme-active site to achieve electrostatic complementarity between the enzyme and substrate (75). Protein engineering, when coupled with detailed stmctural information, is a powerful technique that can be used to alter the catalytic activity of an enzyme in a predictable fashion. 

In 1989, two enzymes based on genetic engineering techniques were introduced, ie, a cloned alkaline protease (IBIS) and a protein engineered Subtihsin Novo (Genencor, California). Lipase and ceUulase types of detergent enzymes have also begun to appear. 

It is likely that any new enzymes isolated by screeners will be quickly and routinely cloned by genetic engineers, and be sequenced and expressed as almost pure proteins. Protein chemists can then evaluate the properties of the new enzyme and determine its three-dimensional stmcture. This vast amount of information allows the protein engineers and their computers to design the enzymes of the future. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

The subtilisin mutants described here illustrate the power of protein engineering as a tool to allow us to identify the specific roles of side chains in the catalytic mechanisms of enzymes. In Chapter 17 we shall discuss the utility of protein engineering in other contexts, such as design of novel proteins and the elucidation of the energetics of ligand binding to proteins. 

Thomas, P.G., Russel, A.J., Fersht, A. Tailoring the pH dependence of enzyme catalysis using protein engineering. Nature 318 375-376, 1985. 

Wells, J.A., et al. Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering. Proc. Natl. Acad. Sci. USA 84 5167-5171, 1987. 

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... 

Enzyme promiscuity is clearly advantageous to chemists since it broadens the applicability of enzymes in chemical synthesis. New catalytic activities in existing enzymes can be enhanced by protein engineering - appropriate mutagenesis of the enzymes [106]. Some of the most illustrative examples of this unusual activity of common enzymes are presented below. 

The biotransformation process has been improved by significant advances in biochemical engineering advances in genetic and protein engineering, microbiological manipulations for the production of enzymes, and the use of biocatalysts in immobilized form and large-scale purification methods. 

Fersht, A. R., Matouschek, A., and Serrano, L. (1992). The folding of an enzyme. 1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771-782. 

Gaertner, H.F., Offord, R.E., Cotton, R., Timms, D., Camble, R., and Rose, K. (1994) Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor. J. Biol. Chem. 269, 7224-7230. 

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