Protein engineering substrate specificity modification

With at least partial understanding of essential features of enzymatic catalysis has come the desire to improve, by the modification of existing enzymes through genetic or chemical means to alter their substrate specificity without loss of catalytic efficiency, the field of protein engineering. A unique feature in the antibody design is that the stereospecific catalysis can be sought for reactions not known to be enzyme catalyzed. 

An interesting feature of DET biosensors is a simple and robust electrode architecture (e.g., no leaking of soluble redox mediators). Additionally, many interfering substances affecting the detection in first- and second-generation biosensors do not interfere with biosensors based on DET. The number of possible analytes is of course restricted to the number of available enzymes, but by use of modem protein engineering techniques the spectmm of analytes will be broadened in future. An example is the modification of the substrate specificity of cellobiose... 

The substrate specificity of an enzyme and its catalytic activity together result from the three-dimensional interaction of the substrate with the side chains of a few nicely positioned amino acids in the protein. Genetic engineering allows the nucleotide sequences coding for these amino acids to be altered as the DNA is transferred from one organism to another. Thus the amino acids which bind the substrate can be altered to accommodate new ones which are sterically excluded from the natural enzyme. The difficulty with this work is not so much the modification of the enzyme, but rather the purification and crystallization which are necessary to solve the three-dimensional structure in the first place. 

The invention of new methods of protein engineering in the past decade has led to the construction of an abundance of P450 mutants with new tailored properties. Recent major achievements include significant increases in productivities, yields, and rates of catalytic turnover as well as modification of substrate specificity and efficient multistep reactions in whole-cell biocatalysts, coming one step closer to the technical application of cytochrome P450 monooxygenases. 

Once enzymes have been modified, they are crystallized and their spatial structure is determined by x-ray crystallography to characterize the structural effects of the specific modifications. Molecular modeling helps to identify which positions in the molecule can be further modified by protein engineering to get a desired effect (specificity and affinity for a substrate, stability at a given pH and temperature). This approach has been used to alter the substrate specificity of subtilisin proteases, to broaden both their pH-activity and pH-stability profiles, and to increase their bleach stability [57]. 

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