TABLE 3

Relative activity of LipT6 variants in 60% methanol

VariantaRelative activity ratiob
Single mutants
    L360F81.2 ± 8.51
    A187F5.3 ± 0.72
    L184F4.5 ± 0.99
    F268Y3.46 ± 0.22
    R215F2.40 ± 0.35
    H154Y1.89 ± 0.25
    L184Y1.51 ± 0.19
    R215Y1.31 ± 0.29
    A187Y1.22 ± 0.43
    F226Y1.08 ± 0.45
    H154W0.94 ± 0.04
    K330Y0.49 ± 0.15
    K330W0.30 ± 0.08
    I11W0.21 ± 0.03
    L360Y0.19 ± 0.09
    L380F0.10 ± 0.05
Double mutants
    A187F/L360F26.5 ± 7.48
    L184F/A187F19.9 ± 0.89
    L184F/L360F0.46 ± 0.05
Triple mutant
    L184F/A187F/L360F0.29 ± 0.06
  • a Each variant was expressed in E. coli, and the soluble cell extract (CE) was used for the screen. SDS-PAGE analysis ensured an appropriate expression level, and hydrolysis activity in buffer ensured no drastic activity loss.

  • b The relative hydrolysis activity of pNPL was calculated as (E/E0)/(E/E0)WT by comparing the activity in the CE from each variant before (E0) and after (E) a 30-min incubation in 60% methanol divided by the same value for LipT6WT (E/E0)WT. The results represent the averages from duplicates.