Saturation mutagenesis

Hydantoinase Enantioselectivity + total activity Inverted enantioselectivity, 5 x increase in total activity Random mutagenesis + saturation mutagenesis + screening E. coli [65]... 

Random mutagenesis + saturation mutagenesis + screening B. subtilis [64]... 

P450gj g from B. megaterium Random mutagenesis Saturation mutagenesis Site-directed mutagenesis UV-vis Mutants showed significantly resistance toward acetone, acetonitrile, dimethylformamide, and ethanol [143]... 

Recently, a method called iterative saturation mutagenesis (ISM) has been proposed and experimentally implemented, which appears to be exceptionally efficient [23,24],... 

Figure 2.3 Schematic illustration of Iterative Saturation Mutagenesis (ISM) involving (as an example) four randomization sites A, B, C and D [23,24].

Figure2.8 BindingpocketofPALfortheacid partofrac-1 showing the geometric position of amino acids 160-163, which were randomized simultaneously by saturation mutagenesis [22],...

The Bacillus subtilis lipase A (BSLA) was the subject of two short directed evolution studies [19,47]. In one case systematic saturation mutagenesis at all of the ISlpositions of BSLA was performed [19]. Using meso-l,4-diacetoxy-2-cyclopentene as the substrate, reversed enantioselectivity of up to 83% ee was observed. In another study synthetic shuffling (Assembly of Designed Oligonucleotides) was tested using BSLA [47]. 

Following epPCR and saturation mutagenesis at hot spots, the o-selective hydan-toinase from Arthrobacter sp. DSM 9771 was converted into L-selective variants. The best L-selective mutant showed a value of 20% ee at about 30% conversion, compared to the WT displaying ee = 40% in favor of the D-methionine-derivative. With the help of an appropriate L-carbamoylase, L-methionine itself was produced. This academic/ industrial effort provided several selective hydantoinases of industrial interest (O May, (Degussa-Hiils), personal communication, 2005). 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

Pseudomonas aeruginosa lipase-catalyzed hydrolysis of racemic ester 23 proceeds with very low enantioselectivity E = 1.1). Sequential use of error-prone PCR, saturation mutagenesis at chosen spots and DNA shuffling resulted in the formation of a mutant whose enantioselectivity was over 50. 

Evans, J., Maccabee, M., Hatahet, Z., Courcelle, H., Bockrath, R., Ide, H. and Wallace, S. (1993). Thymine ring saturation and fragmentation products, lesion bypass, misinsertion and implications for mutagenesis. Mut. Res. 299, 147-156. 

Cephalosporins are a class of antibiotic produced via the intermediate 7-aminocephalos-poranic acid (7-ACA), or 7-aminodesacetoxycephalosporanic acid (7-ADCA). Directed evolution has been used to improve the activity of cephalosporin acylases to produce these intermediates from adipyl-7-ACA or cephalosporin C [68]. Using site-directed saturation mutagenesis and a selection system whereby the E. coli host is dependent on leucine liberated from derivatives of the cephalosporin side-chains, a mutant was found that increased the catalytic efficiency toward adipyl-7-ADCA by 36-fold. 

DeSantis, G., Wong, K., Farwell, B. et al. (2003) Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). Journal of the American Chemical Society, 125, 11476-11477. 

Wong, T.S., Tee, K.L., Hauer, B. and Schwaneberg, U. (2004) Sequence saturation mutagenesis (SeSaM) a novel method for directed evolution. Nucleic Acids Research, 32, e26. 

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... 

Reetz, M.T., Carballeira, J.D. and Vogel, A. (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angewandte Chemie (International Edition in English), 45, 7745-7751. 

Hill Eubank, D., Burstein, E., Spalding, T., Brauner-Osborne, H. and Brann, M. Structure of a G-protein coupling domain of a muscarinic receptor predicted by random saturation mutagenesis./. Biol. Chem. 271 3058-3065,1996. 

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