Genetic engineering: of enzymes

Genetic engineering procedures Microbe genetic engineering Plant genetic engineering of enzymes, 12 65 of Pseudomonas, 12 476 of silk, 22 633... 

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. 

Figure 8. Genetic engineering of enzymes for hydrolysis of esters...

Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. 

Lee, J. M. and G. An, "Industrial application and genetic engineering of plant cell cultures", Enzyme Microb. Technol. 8 (1986) 260 - 265. 

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushel ME, Sanders PG (1995), Genetic engineering of hybridoma glutamine metabolism, Enzyme Microb. Technol. 17 98-106. 

Forest Products Laboratory have discovered a xylose-fermenting yeast (Candida tropicalis). Thus it now is possible to convert all wood sugars to ethyl alcohol with a combination of yeasts. Isolation of the specific enzymes and genetic engineering of the enzymes could dramatically improve the efficiency of this conversion. 

In our view, genetic engineering of future enzymes for industrial uses should consider not only their catalytic properties, but also their potential for isolation and immobilization. Designing enzymes to allow selective, high affinity immobilization by adsorption on a relatively inexpensive matrix should greatly increase the attractiveness of enzyme bioreactor processes. 

The genetic engineering of qinghao (4. annua) has also been paid great attention recently some preliminary results about the early stage of qinghaosu biosynthesis have been reported. For example, amorpha-4,11-diene synthase, an enzyme responsible for the cyclization of farnesyl diphosphate into ring sesquiterpene, has been expressed in Escherichia coli and production of amorpha-4,11-diene (122) was identified. °... 

Genetic engineering of hen egg-white lysozyme has been used by Kirsch el al. (1989) as an approach to studying the structure—function relationships of lysozyme. Thus, they offer evidence from site-directed mutagenesis of cloned lysozyme (expressed in yeast), that Asp-52 and Glu-35 are vital for the expression of lysozyme. However, it is curious that conversion of Asp-52 to the amide resulted in a form of the enzyme that still had 5% of the normal activity. Conversion of Glu-35 to the amide, on the other hand, resulted in a lysozyme that was devoid of all activity. It was demonstrated by mutagenesis of Asp-IOI to Gly that the ionization of this residue contributes thermodynamically to the association of lysozyme with the inhibitor chitotriose. 

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