Site saturation mutagenesis

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. 

Parikh, M.R. and Matyoumara, I., Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of / -fucosidase. J. Mol Biol, 2005, 352, 621-628. 

Gene Site Saturation Mutagenesis (GSSM) All single amino acid substitutions explored. Technically out of reach for most researchers. US Patent 6171820... 

CAST combinatorial active-site saturation mutagenesis test... 

Padhi, S.K., Bougioukou, D.J., and Stewart, J.D. (2009) Site-saturation mutagenesis of Tryptophan 116 of Saccharomyces pastorianus old yellow enzyme uncovers stereocomplementary variants. J. Am. Chem. Soc., 131, 3271-3280. 

Site saturation mutagenesis methods and appbcations in protein engineering. Biocatal. Agric. Biotechnol, 1,181—189. 

M., Salcuma, M., Mansell, D Stephens, G. M., Gardiner, J.M., and Scmtton, N.S. (2011) A site-saturated mutagenesis study of pentaeryihritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity. ChemBioChem, 12, 738-749. 

Wu, W.H., Zhu, D.M., and Hua, L. (2009) Site-saturation mutagenesis of formate dehydrogenase from Candida boidinii creating effective NADP -dependent FDH enzymes./. Mol. Catal. B., 61, 157-161. 

Computational enzyme-substrate docking studies carried out on a CRED from Sporoholomyces salmonicolor showed that residues Met 242 and Gin 245 were in close proximity to the para-substituent of acetophenones in the substrate binding site. Site-saturation mutagenesis of Met 242 or Gin 245 (replacing these residues with all 19 other possible amino acids) and double mutation of both Met 242 and Gin 245 were carried out to enhance the enzyme s enantioselectivity toward the reduction of para-substituted acetophenones. The enantiopreference was inverted from R)- to (S)-configuration for three Gin 245 mutants that were obtained. Preference for the formation of (S)-enantiomeric alcohols was shown for four Met 242 mutant enzymes when compared to the wild-type enzyme Gin 245 and Met 242/Gln 245 double variations inverted the enantiopreference and affected the enantiomeric purity of the product alcohols. Residues 242 and 245 also exerted an effect on the catalytic activity of this GRED [23]. 

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