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Home >>Protein Engineering, Immunotoxins and Drug Designing - Engineering of Macromolecules >> Rationale of Protein Enzyme Engineering
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Rationale of Protein Enzyme Engineering - Although thousands of proteins have been characterized in prokaryotes and eukaryotes, only few became commercially important. This is due to the high cost of isolating and purifying enzymes in sufficient quantities.

Although the cost aspect has been overcome by producing an enzyme in large quantities in bacteria, for its industrial application, an enzyme (outside the cell) should also have some characteristics in addition to those of enzymes in the cells. These characteristics may include the following:
(i) enzyme should be robust with a long life;

(ii) enzyme should be able to use the substrate supplied in the industry even if it differs slightly from that in the cell;

(iii) enzyme should be able to work under conditions (e.g. extremes of pH, temperature and concentration) of the industry even if they differ from those in the cell.

In view of the above, enzyme should be engineered to meet the altered needs. Therefore, efforts have been made to alter the properties of the enzymes. Following is the list of properties that one needs to alter in a predictable manner for protein or enzyme engineering.

(1) Kinetic properties of enzyme turnover and Michaelis Constant, Km.
(2) Theremostability and the optimum temperature for the enzyme.
(3) Stability and activity of enzyme in nonaqueous solvents.
(4) Substrate and reaction specificity.
(5) Cofactor requirements.
(6) Optimun pH.
(7) Protease resistance.
(8) Allosteric regulation.
(9) Molecular weight and subunit structure.

For a particular class of enzymes, variation in nature may occur for each of the above properties, so that one may like to combine the optimum properties to get the most efficient form of the enzyme.

This aspect of protein engineering will be illustrated using the example of glucose isomerases, which convert glucose into other isomers like fructose and are used to make high fructose corn syrup vital for soft drink industry. It exhibits wide variation in its properties.
Sometimes, it may not be possible to get a combination of optimum properties. For instance, an enzyme with highest activity may not be the most stable. Therefore, a compromise in properties may have to be made, if we have to select an enzyme from the available variability or even if we create variability by mutagenesis.
However, if structure and function relationship of an enzyme is known, the structural features for desirable function may be combined and protein engineering techniques may then be used to create a novel enzyme exhibiting a combination of all desirable functional properties.

Glucose isomerase belongs to a TIM barrel family of enzymes which resemble each other in having a highly characteristic domain called TIM barrel, with active site for catalytic action at one end. This TIM barrel may be found in enzymes that may differ in sequences and may catalyze different reactions.
As earlier discussed, since similarities of structure of protein meant similarities in function, TIM barrel presents a challenge to this concept. However, it is curious tbat some enzymes in this family appear in pairs in their metabolic pathways so that they catalyse two consecutive steps thus showing coupling of their functions.
As an example of two enzymes of TIM barrel family, while 'triose phosphate isomerase' is one of the most efficient catalysts, 'glucose isomerase' is relatively very inefficient.
Therefore, if 'glucose isomerase' enzyme is redesigned to use the highly efficient domain of TIM barrel family, it will be a remarkable achievement for soft drink industry. Efforts in this direction are being made (see later for methods of protein engineering).
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