Directed random mutagenesis

Fromant, M., Blanquet, S., and Plateau, P. (1995). Direct random mutagenesis of genesized DNA fragments using polymerase chain reaction. Analytical Biochemistry 224, 347-353. 

Directed evolution of enzymes has been used to improve the reducing function of the enzymes. For example, this method was used to eliminate the cofactor requirement of B. stearothermophillus lactate dehydrogenase, which is activated in the presence of fructose 1,6-bisphosphate [12]. The activator is expensive and representative of the sort of cofactor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant that is almost fully... 

Eggert, T., Jaeger, K.-E. and Reetz, M.T. (2004) Directed evolution of random mutagenesis a critical evaluation, in Enzyme Functionality (ed. A. Svendsen), Marcel Dekker, Inc., New York, pp. 375-390. 

There have been many attempts to improve protein stability and protein properties, utilizing methods such as random mutagenesis, directed evolution, and rational protein design approaches. In general, these methods are far from straightforward and can be time-consuming. In addition, the stabilization of proteins without loss of function is not a trivial problem. 

The sequence of chapters mirrors the steps in a standard directed-evolution experiment. In the beginning, various methods for the creation of molecular diversity are considered. S. Brakmann and B.F. Lindemann (Chapter 2) present protocols for the generation of mutant libraries by random mutagenesis. Two chapters deal with the particularly powerful approach of in-vitro recombination. H. Suenaga, M. Goto, and K. Furukawa (Chapter 3) describe the application of DNA shuffling, and M. Ninkovic (Chapter 4) presents DNA recombination by the S tEP method. 

Therefore tailoring the enzyme to the processing conditions would be preferred recent advances in random mutagenesis and directed evolution, notable features conceptually illustrated in Figure 11.3, offer the possibility of achieving this goal in the absence of detailed structural information on the protein. 

The carbon-carbon forming ability of aldolases has been limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has most often not produced the desired effect. Directed evolution approaches have furnished novel activities through multiple mutations of residues involved in recognition in no instance has a key catalytic residue been altered while activity is retained. Random mutagenesis resulted in a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile (Wymer, 2001). 

Fig. 1. A working strategy for directed enzyme evolution. The screening method should ensure that small enhancements brought about mainly by single mutations can be measured. The evolution of a new, useful enzyme requires an effective strategy for accumulating many such small improvements. Beneficial mutations can be accumulated in a sequential generations of random mutagenesis and screening or b by (random) recombination...

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