Directed evolution of a thermostable esterase

Abstract

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Figure 1

Activities of purified wild-type (WT) and evolved esterases. (A) Evolutionary progress of the melting temperatures (T_m) and specific activities of evolved esterases. DSC was performed at least twice on each purified enzyme. (B) Activities of WT (•), and evolved esterases 1A5D1 (■), 2A12 (□), 3H5 (⋄), 4G4 (○), 5H3 (▴), and 6sF9 (▿), as a function of temperature. The temperature of optimal activity increases with increasing thermostability.

Figure 2

Results of screening 1,100 clones picked from the second-generation library. Values from multiple screens of the parent enzyme (1A5D1) fall within the circle. Variants with less than 20% of the initial activity of 1A5D1 have been removed.

Figure 3

Amino acid mutations in the most thermostable variants from each generation (∗ indicates a new mutation). With the exception of 6sF9, which was generated by DNA shuffling of five parents, all variants were generated by random mutagenesis of a single parent.

Figure 4

Model of pNB esterase constructed based on homology to esterases of known structure (18), showing positions of thermostabilizing mutations (blue). The model is displayed using molscript (31). Active-site residues are shown in red. Residues 1–312 are shown in green. Thermostabilizing mutations are clustered in the C-terminal domain.