Enzyme Engineering Strategies

MW 27,500) with no cofactors or metal ions reqnirement for its function, it displays Michaelis-Menten kinetics and it is secreted in large amounts by a wide variety of Bacillus species. Subtilisin is also among the most important industrial enzymes due to its use in laundry detergents. Protein engineering strategies for subtilisin have focused on a number of aspects, namely catalysis, substrate specificity, thermal and oxidative stability and pH profile. We will describe briefly each of these aspects. 

The last examples demonstrate that attention has shifted away from singlegene engineering strategies and towards more complex approaches involving the simultaneous overexpression and/or suppression of multiple genes. The use of regulatory factors to control the abundance or activity of several enzymes is also becoming more widespread. 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

RD Chen. A general strategy for enzyme engineering. Trends Biotechnol 17 344— 345, 1999. 

Enzyme engineering will increasingly drive the development of robust enzymes tailored to specific bioprocesses. The strategies used for improving the stability of enzymes will continue to focus on the generation and screening of libraries of mutants produced by random or targeted amino acid substitutions, however, alternative approaches also include... 

This chapter focuses on biocatalytic methods that have been demonstrated in the aldol and related reactions. Additionally strategies for enzyme engineering and future challenges faced in this field are addressed. 

Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH. Evolving strategies for enzyme engineering. Curr. Opin. Struc. Biol. 2005 15 447-452. 

Combining site-directed mutagenesis strategies with chemical modification is a popular tool in both enzyme engineering and mechanistic studies. This has often been applied to the subtilisin from Bacillus lentus (SBL), or savinase. Subtilisins are... 

With directed evolution we can engineer enzyme properties rapidly and with a high probability of success. Many enzymes that have been improved by directed evolution are listed in Tab. 4-3. This powerful biocatalyst engineering strategy creates new opportunities in organic synthesis new and improved bioconversion processes can be developed and novel compounds that are otherwise inaccessible by classical chemistry can be synthesized. In addition, the molecules created by directed evolution offer an excellent opportunity for improving our still poor understanding of sequence-structure-function relationships. 

Enzyme engineering is undergoing the most profound and exciting transformation in its history and promises unprecedented expansion in the scope and applications of modified or improved enzymes with desired physical and catalytic properties. Two complementary strategies are currently available rational design and directed evolution. Although both approaches have been met with great success, each has its limitations (13). 

Regarding to the H6H enzyme, Liu et al. [103] reported the cloning, expression in E. coli and characterization of the enzyme isolated from hairy roots of A. tanguticus. This report has focused on the functional studies of the H6H enzyme with the aim to improve alkaloids production in plants by the application of metabolic engineering strategies [103],... 

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