Site-Directed Mutagenesis as Applied to Biocatalysts

Bicyclo[3.2.0]carbocyclic Molecules and Redox Biotransformations: The Evolution of Closed-Loop Artificial Linear Biocatalytic Cascades and Related Redox-Neutral Systems

“…Consistent throughout these in vitro studies has been a reliance on the biocatalytic activity of native enzymes. Despite the concurrent emergence of site-specific mutagenesis as a powerful genetic engineering technique to promote site-specific changes to enzymes, it has made little relevant impact on the biocatalysis of bicyclo[3.2.0]carbocyclic molecules by YADH, HLADH or HSDH [130]. The original methodology was introduced in the early 1980s [131][132][133], but was replaced by a much simpler protocol developed several years later by Kunkel [134], thereafter prompting studies on various ADHs.…”

Biotechnological relevance of the lipase A from Candida antarctica

“…In this regard, the first directed evolution of CALA was performed employing a combinatorial active-site saturation test (CAST), in such approach, pairs of amino acid residues surrounding the catalytic site of an enzyme are chosen for complete randomization [36,146,147]. As a result, iterative rounds of CASTing yielded variants with good selectivity towards both the (R)-and (S)-4-nitrophenyl 2-methylheptanoate; indeed, the best enzyme variants had an enantiospecificity values of 52 (S) and (R), while wild-type CALA has a modest E-value of 5.1 [148].…”

“…while the potential of site-directed mutagenesis and dynamic simulations increase every day [35][36][37]. Mutagenesis of the active center may permit to modify the active site (e.g., replacing a Ser residue with a metal) therefore creating new enzyme activities [38].…”