TABLE 1

Selected mutants of preαproK-AAO libraries

VariantLibraryMutationLocationSecondary motifTotal activity improvement (fold)aT50 (°C)
FX7M-I-IICACH91NAACFAD-binding domainLoop9664.3
13H2M-IVATAI194VGTASubstrate-binding domainLoop4.960.8
10G5M-IVTTGL170 MATGSubstrate-binding domainAlpha helix4.960.5
7A11M-IIGATD341NAATSubstrate-binding domainAlpha helix4.659.3
4C7M-IIAGAR481SAGTSubstrate-binding domainLoop4.561.5
12G12M-IACAT[50K]AGCA AGCS88TACCSignal peptide; FAD-binding domainLoop1.961.8
PreαproK-AAO (parental type)158.8
EcAAO47.5
  • a The total activity improvement over parental type was estimated with 2 mM p-methoxybenzyl alcohol as the substrate in 100 mM phosphate buffer (pH 6.0).