Enzymatic reactions genetic engineering

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. 

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. 

A further compHcation remains in the control of enzyme activities which to a large extent is dependent upon expression during the fermentation. One potential solution is to add supplementary enzymes to the cell-free extract. Such a precedent has already been set by a few reported cases where whole cells were mixed with the isolated enzyme for ex vivo cofactor recycle. E>espite these problems, there is no doubt that as genetic engineering for expression of the desired enzyme is improved, more systems wiU be tested in the cell-free environment [26]. At the very least it is clear that cell-free extracts, combined with network topology analysis can provide an excellent basis for effective analysis and targeting of the network so as to insulate the desired pathway from undesired enzymatic reactions [27]. 

Aim of this research was to increase efficiency of producing T. thermophilus PyrNPase in cells of genetically engineered E. coli strain by optimizing the structure of the respective translated mRNA and to investigate enzymatic synthesis of purine 3 -fluoro-3 -deoxy- and 3 -fluoro-2, 3 -dideoxynucleosides possessing antiviral and cytostatic activities from the available pyrimidine nucleosides engaging tandem reactions in the presence of recombinant nucleoside phosphorylases [8],... 

Other possible syntheses are based on a plas-tein reaction (cf. 1.4.6.3.2) with an N-derivatized aspartic acid and phenylalanine methylester or on bacterial synthesis of an Asp-Phe polymer, achieved by genetic engineering techniques, enzymatic cleavage of the polymer to Asp-Phe, followed by acid or enzyme catalyzed esterification of the dipeptide with methanol. 

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