YqjM mutants

Under the standardized screening conditions, WT YqjM displays only about 3% conversion in the model reaction and moderate enantioselectivity (76% ee) in favor of (J )-8a, which indicates that the substrate is not well accepted. Therefore, in order to evolve active and stereoselective YqjM mutants, screening was focused primarily on activity. Accordingly, mutants were identified by automated gas chromatography (GC) and those active above the WT threshold were checked for enantioselectivity. Since this involved the optimization of two distinct catalytic parameters, the quality of each hit was defined by two numbers, namely, % conversion (0-100%) and % ee for the (J )- or (S)-regime, respectively [37]. Then the total impact (TI) was calculated by multiplying the two parameters (Table 5.5). Such a procedure leads to the... 

Table S.S Evaluation ofthe active YqjM mutants identified in the first round of saturation mutagenesis/screening with 7a— 8a as the model reaction [37],...

Figure 5.8 A total of 20 amino add residues lining the binding pocket ofYqjM, mainly above the flavin, were addressed by saturation mutagenesis (CAST, single-residue sites with NNK codon degeneracy) [37], Residues with a star did not lead to improved mutants. Cartoon model of YqjM from PDB entry 1Z44 is illustrated (Chain B, white Chain A, green red...

Figure 5.10 Pathways leading to active YqjM the second round. The first-generation mutants having maximum enantioselectivity templates and resulting second-generation [37]. Dashed lines and light gray boxes indicate mutants are isochromatic. the first round of mutagenesis, and dotted lines...

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