The introduction of the techniques of modern molecular biology, beginning in the 1970s, have made possible the genetic engineering of proteins. New proteins can be created and existing proteins altered. Enzymes are proteins that function in chemical reactions by making the reaction occur more easily than if the enzyme were absent.
Enzymatic engineering includes a wide variety of techniques related to the manipulation of molecules to create new enzymes, or proteins, that have useful characteristics. These techniques range from the modification of existing enzymes to the creation of totally new enzymes. Typically, this involves changing the protein on a genetic level by mutating a gene. Other ways that modifications to enzymes are made are by chemical reactions and synthesis.
Genetic engineering can improve the speed at which the enzyme-catalyzed chemical reaction proceeds, and can improve the amount of product that is generated by the chemical reaction. Furthermore, an enzyme that has a desirable activity, but which operates very slowly in its parent organism, can be taken out of that organism and installed in another organism where it performs faster. An example of this is the speeding up of the enzyme penicillin-G-amidase by slicing the enzyme into the genetic material of Escherichia coli.
Enzymes are relatively large, complex molecules that catalyze biochemical reactions. These include such diverse reactions as the digestion of sugars, replication of deoxyribonucleic acid (DNA), or combating diseases. For this reason, the creation of improved enzymes is an important field of study. A variety of steps are involved in the creation of modified enzymes. These entail a determination of the molecular structure, function, and modifications to improve the effectiveness.
The key to creating new and improved enzymes and proteins is determining the relationship between the structure and function of the molecule. The structure of an enzyme can be described on four levels: The primary structure is related to the sequence of amino acids that are bonded together to form the molecule. The amino acids that make up the enzyme are determined by the DNA sequence of the gene that codes for the enzyme. When enough amino acids are present, the molecule folds into secondary structures known as structural motifs. These are further organized into a three-dimensional, or tertiary structure. In many enzymes, multiple tertiary structures are combined to give the overall quaternary structure of the molecule. Scientists have found that the specific structural organization of amino acids in an enzyme are directly related to the function of that enzyme.
Structural information about enzymes is obtained by a variety of methods. The amino acid sequence is typically found using DNA sequencing. In this method, the pattern of nucleotides is determined for the gene that encodes the enzyme. This information is then compared to the genetic code to obtain the amino acid sequence. Other structural information can also be found by using spectroscopy, chromatography, magnetic resonance, and x-ray crystallography.
To investigate the function of an enzyme, it is helpful to be able to create large quantities of it. This is done by cloning a desired gene. Polymerase chain reaction (PCR), which is a method of making a large number of copies of a gene, is particularly helpful at this point. The cloned genes are then incorporated into a biological vector such as a bacterial plasmid or phage. Colonies of bacteria that have the specific gene are grown. Typically, these bacteria will express the foreign gene and produce the desired enzyme. This enzyme is then isolated for further study.
Restriction enzymes— Enzymes that recognize certain sequences of DNA and cleave the DNA at those sites. The enzymes are used to generate fragments of DNA that can be subsequently joined together to create new stretches of DNA.
Site directed mutagenesis— The alteration of a specific site on a DNA molecule. This can be done using specialized viruses.
To create new enzymes, the DNA sequence of the gene can be modified. Modifications include such things as deleting, inserting, or substituting different nucleotides. The resulting gene will code for a slightly modified enzyme, which can then be studied for improved stability and functionality.
One of the major goals in enzymatic engineering is to be able to determine enzyme structure and functionality based on the amino acid sequence. With improvements in computer technology and our understanding of basic molecular interactions, this may someday be a reality.
Another promising area of enzymatic engineering is the engineering of proteins. By designing or modifying enzymes, the proteins that the enzymes help create can also be changed, or the amount of the protein that is made can be increased. Also, since enzymes are themselves proteins, the molecular tinkering with protein structure and genetic sequence can directly change the structure and function of enzymes.
Laskin, A.I., G. Li, and Y.T. Yu. “Enzyme Engineering.” Annals of the New York Academy of Science 864 (December 1998): 1–665.
London South Bank University, Faculty of Engineering, Science, and the Built Environment. “Enzyme Engineering” <http://www.lsbu.ac.uk/biology/enztech/engineering.html > (accessed November 24, 2006).
Swiss Federal Institute of Technology, Zurich. “Enzyme Engineering” <http://www.chab.ethz.ch/publicrelations/publikationen/chimia/f10.pdf> (accessed November 24, 2006).