Investigating the activity and thermophysical properties of an ancestral sequence reconstructed aldolase

Sammanfattning

Biocatalysts are an important implementation to achieve green chemistry. However, producingsufficient biocatalysts for an industrial setting can be haltered by enzymes specificity to theirnative substrates. Ancestral sequence reconstruction could potentially aid biocatalyst synthesis,when enzyme evolution is not applicable, by producing a more promiscuous enzyme as astarting point for engineering. Aldolases are desirable enzyme to utilize in biocatalysis becauseof their ability to facilitate new carbon-carbon bonds. Therefore, this work investigated anancestral sequence reconstructed aldolase and four mutated variants propensity to catalyse nonnative aldolase reactions of the extant fructose-6-phosphate aldolase to understand if thetechnique is a useful first step in the workflow of producing a biocatalyst. Moreover, the generalthermophysical properties of this ancestral aldolase was investigated. Neither the ancestralsequence reconstructed aldolase nor its mutants exhibited any substantial improvement withnon-native substrates. Surprisingly, the produced mutants mirroring previously producedfructose-6-phosphate aldolase mutants exhibited different effects on the investigated reactions.Most interestingly, one of the ancestral aldolase mutants was capable of catalysis after heattreatment at 100 °C. Thereby exhibiting higher thermal stability than the extant fructose-6-phosphate aldolase.