Enzymes mutagenesis methods

Entry Enzymes Mutagenesis Methods Screening Results References... 

Systematic experimental and theoretical studies of this kind are helpful in performing in vitro evolution of enantioselectivity. Nevertheless, several questions are not fully answered. Are remote mutations more important than those close to the active site, or is the opposite true Is it more effective to allow randomization all over the enzyme rather than focusing on the region around the active site (or vice versa) To be sure, when applying epPCR or any other mutagenesis method that more or less... 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

Plapp, B. V. (1995) Site-directed mutagenesis A tool for studying enzyme catalysis, Methods Enzymol. (Enzyme Kinetics and Mechanism, Part D) 249, 91-199. 

A subsequent report describes the evolution of the same enzyme for the hydrolysis of 3-phenylbutyric acid resorufin ester using both a mutator strain and error-prone PCr(2°7] Mutants were screened for improved enantioselectivity based on a microtiter plate assay using the optically pure R- or S-esters. Both mutagenesis methods generated first-generation mutants with higher enantioselectivity ( =6.6 and 5.8 compared to wild-type E=3.5). 

This enzyme assay method was recently used to kinetically characterize a synthetic enzyme, isomelezitose synthase, which was prepared by site-directed mutagenesis of sucrose isomerases. Continuous measurement of fluorescence ehanges in a 384-well plate, containing 4,4 -o-BBV/HPTS and isomelezitose synthase, allowed these authors to measure the concentration of fructose and isomaltulose in real time and to kinetically follow the conversion of sucrose into fructose and isomaltulose. ... 

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