Mutagenesis, random

Cunningham, B.C., Wells, J.A. Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure. Prot. Eng. 

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... 

Parallel to the modification of the catalytic performance in Baeyer-Villiger oxidations, random mutagenesis was successfully applied to improve the stereoselectivity of CHMOAcineto hi cascs of essentially racemic sulfoxide formation. In addition, enantiodivergent clones with >98% ee for both antipodal products were identified (Table 9.5) [205]. However, improvement in stereoselectivity of mutant enzymes was often accompanied by increased formation of sulfone. This effect can also be utilized to resolve racemic sulfoxides. 

Table 9.5 Random mutagenesis of CHMO, 6to toward enantiodivergent sulfoxidations. ...

Using a random mutagenesis approach, respiratory-deficient (34) and temperature-sensitive (46, 47) mutants of the Rieske protein of the yeast bc complex have been selected. A large fraction of the point mutants had changes of residues in the bottom of the cluster binding subdomain (the loop /S7-/38) and in the Pro loop comprising residues 174-180 of the ISF (Fig. 9 see Section III,B,3) this indicates the importance of the Pro loop for the stability of the protein. Amino... 

Section 5.3.2.3) combined with over-expression of IPPI resulted in enhanced astaxanthin accnmnlation to 1.4 mg/g dry cell weight (DCW). Further increases to 45 mg/g DCW were obtained by random mutagenesis of GGPPS, perhaps by altering enzyme response to substrate-level feedback inhibition. 

Barriault D, M Sylvestre (2004) Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in Region III. J Biol Chem 279 47480 7488. 

Another example from the "bio-world" is the production of micro-organisms for optimum product yield and quality. An example could be micro-organisms for the production of penicillin, whereas recently there have been developments to explore routes to produce monomers for synthetic polymers by means of micro-organisms. Diversity in the micro-organisms to be tested can be achieved either by genetic engineering or by random mutagenesis. 

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. 

Figure 3.1 Overview of DNA library creation strategies. Random mutagenesis introduces mutations at positions throughout the gene sequence. Semi-rational design randomizes only the specific position(s) of interest. Gene shuffling brings existing sequence diversity from different parental DNA sequences together to form a chimeric library...

Leung, D.W., Chen, E. and Goeddel, D.V. (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1, 11-15. 

Seng Wong, T., Roccatano, D., Loakes, D. et al. (2007) Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+) a random mutagenesis method with consecutive nucleotide exchanges that complements the bias... 

We have employed a more systematic random mutagenesis approach by dividing the BDI into consecutive clusters of 10 amino acid residues (Boxes) that are individually replaced with a string of 10 alanine residues in the full-length Hisg-tagged allele to facilitate affinity purification of the mutant protein (Valasek et ah, 2004). Alternatively, clusters rich in... 

DsRed (Fig. 5.3E) is a bright RFP with excitation and emission maxima at 558 and 583 nm, respectively. Despite the bright red fluorescence, application of DsRed has been restricted, because of slow and inefficient maturation and its tetrameric structure [70, 71], The poor maturation efficiency has been overcome by random mutagenesis, which resulted in the fast maturing variant DsRedTl [72]. However, DsRedTl remains tetrameric. 

TurboFP was also used as a basis for far-red fluorescent proteins (fRFPs). Residues surrounding the chromophore were mutagenized to create a library, which was subsequently subjected to random mutagenesis. A bright far-red variant with excitation and emission maxima at 588 and 635 nm, respectively was isolated and named Katushka [79]. This fast-maturing protein has an... 

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