Thioesterases in fruit and the generation of high impact aroma chemicals

Abstract

Volatile organosulphur compounds (VOSCs) are key ingredients in the aroma of tropical fruit where they are active as both free thiols and the respective thioesters. Present as trace high-impact flavourings, VOSCs are however problematic to extract and have therefore become targets for bioproduction. This work focuses on the endogenous thioesterases in tropical fruit believed to catalyze the liberation of thiol VOSCs from thioester precursors. Using a simple and sensitive colourimetric assay, a thioesterase activity toward VOSCs was identified in purple passion fruit (Passiflora edulis Sims). The enzyme was identified as a cell wall-bound protein in the mesocarp of the fruit. Following extraction with salt solutions, the thioesterase was purified 150-fold and shown to be associated with a 43 kDa polypeptide. Affinity labelling with a biotinylated fluorophosphonate suicide probe showed the enzyme to be a serine hydrolase, with MS-MS sequencing of tryptic digests identifying it as a pectin acetylesterase (PAE). Putative thioesterase PAEs were subsequently cloned from passion fruit and Arabidopsis thaliana. The observation that an esterase involved in cell wall modification had a secondary role in hydrolysing esterified VOSCs led to the consideration of further fruit species as a source of the enzyme. Orange (Citrus sinensis) was particularly abundant in thioesterase activity. The enzyme was purified 85-fold and identified as a homologous 43 kDa basic (pi: 9) PAE. The enzyme was stable (t(_1/2): 7 days 22 hours) and demonstrated a high turnover toward VOSCs (k(_cat): 7.85 sec(^-1)). Freeze-dried orange peel was found to retain activity (>90% activity, 3 months 4 C) and demonstrated comparable productivities to those of immobilized microbial enzymes. Here we have initiated a programme for developing processes for the bioproduction of VOSCs, in which the potential of plant glycohydrolases has been demonstrated.