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. 2020 Oct 15;5(43):27896–27908. doi: 10.1021/acsomega.0c03070

Table 2. HNL Productivity, Activity, and Enantiomeric Excess of Selected PlamHNL Variantsa.

variants dAffinityb (kcal mol–1) productivity (U L–1) specific activityc (U mg–1) % eefd
N85H –0.9664 477 402 ± 36 85
L98W –0.7173 N.D. N.D. N.D.
L98E –0.6709 N.D. N.D. N.D.
N85Y –0.6488 233 304 ± 18.2 92
R58W –0.5245 N.D. N.D. N.D.
W109H –0.4386 160 205 ± 14.2 22
A74W –0.4163 N.D. N.D. N.D.
N85E –0.3178 382 290 ± 7.6 53
R58K –0.2335 N.D. N.D. N.D.
N85Q –0.2192 180 165 ± 26.7 15
Y60A –0.1977 N.D. N.D. N.D.
Y124A –0.1061 N.D. N.D. N.D.
T95A –0.0494 580 695 ± 42 86
N85H/T95A   450 420 ± 8.6 81
N85Y/T95A   457 401 ± 8.5 88
wild-type   420 506 ± 7.8 76
a

All of variants were selected based on a calculation of the alanine or residue scanning functionality around 14 amino acid residues in the binding pocket using the MOE program to identify critical residues for affinity. N.D. = not detectable, which indicates the case that the measuring enzyme activity was the same as omitting the enzyme reaction (control).

b

dAffinity values refer to the relative binding affinity of the mutation to the wild-type protein. dAffinity is equal to the Boltzmann average of the relative affinities of the ensemble. A more negative value indicates a mutation with better affinity.

c

Specific activity was assayed after 5 min in a reaction mixture containing 2-chlorobenzaldehyde (50 mM) and KCN (100 mM) at 25 °C with the same amount (0.64 μg) of purified PlamHNL variants.

d

Enantiomeric excess was obtained for (R)-2-chloromandelonitrile synthesis by HPLC with the same concentration of purified PlamHNL variants (1.2 Unit).