Enzymatic genetically engineered enzymes

By means of genetic engineering, including cloning and site-directed mutagenesis, it has become possible for modern synthetic chemists to utilize a sufficient amount of isolated enzyme catalysts and to modify the reactivity, stability, or even specificity of enzymes. Therefore, polymerizations catalyzed by isolated enzyme are expected to create a new area of precision polymer syntheses. Furthermore, enzymatic polymerizations have great potential as an environmentally friendly synthetic process of polymeric materials. 

In vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

Genetic engineering nowadays allows us to control gene expression independent of the natural inducers of enzymatic activity and independent of the natural host that once harboured the enzyme. This is especially relevant since natural isolates are specialized in degradation of the educt, not synthesis of a product. Frequently, efficient degradation of the desired product is observed, which can be prevented by using a host different from the natural isolate. 

Researchers at Ajinomoto have used genetic engineering in a directed fashion to improve yields of amino acids produced by microbial fermentations [31]. For example, by increasing the amount of the enzyme for the step labeled 1 in Fig. 4, they were able to increase the final concentration of threonine in a batch cultivation from 17.5 to 25 g/liter. Subsequent amplification of the enzymatic activity for step 2 in Fig. 4 in concert with the amplification of step 1 gave further yield enhancement to 33 g/liter of culture. 

Hydrophilicity is an important criterion for the use of synthetic polymers. Existing methods for surface modihcation of synthetic hbers are costly and complex. Therefore, the enzymatic surface modihcation of synthetic hbers is a new and green approach to synthesize polymers with improved surface properties. Use of enzymes for surface modihcation of polymers will not only minimize the use of hazardous chemicals but also minimize the environment pollution load. Besides these, the enzyme-modihed polymers can also immobilize those enzymes which can only bind to the selective functional groups present on the polymeric surface such as —COOH and —NH2. Similarly, substrates can immobilize on the solid matrix (or polymer), which will be easily accessible to the enzymes. Genetic engineering can be employed for the modihcation of active sites of enzymes for better polymer catalysis. 

The industrial scale-up of enzymatic technology is both highly complicated and expensive. Moreover, a primary regulatory hurdle will involve demonstrating that the end products of the cholesterol-enzyme reaction and the novel compounds formed through genetic engineering are harmless. 

In yeast and mycelial fungi, xylose is metabolized via coupled oxidation-reduction reactions . Xylose reductase is the enzyme involved in the reduction of xylose to xylitol. Sequential enzymatic events, through the oxidation of xylitol to xylulose, lead to the utilization of xylose. Many yeast species utilize xylose readily, but the ethanol production capability is very limited. Only a few yeast species effectively produce ethanol from xylose these include Pachysolen tan-nophilus, Candida shihatae and Pichia stipitis [80]. The production of ethanol from xylose by these three yeast strains has been studied extensively in recent years. Recently, genetically engineered yeast strains have been constructed for more effective conversion of xylose to ethanol. 

Enzymes have high potential in organic synthesis. Applications have been until now mainly based on kinetic resolution, but many opportunities exist to use enzymatic catalysis for enantioselective syntheses. Recently, it was shown by Reetz et al. that a combination of genetic engineering and mutagenesis can easily provide modified enzymes of greatly improved stereoselectivity for the transformation of a given substrate [114]. This concept should find wide applications in catalyzed enantioselective reactions. 

In spite of the progress that has been made, several difficulties limit the use of cell-free enzymes for the synthesis of polysaccharides. The major problem is the complexity of many polysaccharide-synthesizing systems. Isolation, purification, and stabilization of the required enzymes is often difficult, as many enzymes lose activity when they are no longer membrane-associated. Enzyme isolation from eukaryotic sources is tedious, because of low cellular enzyme concentration. It is unlikely that cell-free enzymatic synthesis will provide better routes to most natural polysaccharides than do fermentation and isolation. The use of genetic engineering,... 

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