Cloning and secretion of tomato hydroperoxide lyase in Pichia pastoris

Hydroperoxide lyase (HPL) gene from tomato leaves was isolated and comprised of 1431 nucleotides encoding a protein of 476 amino acids, corresponding to a molecular mass of 53,480 Da. The protein was expressed in Pichia pastoris as a secreted enzyme (rLeHPL) after 24 h of culture incubation and induction with methanol. However, no intracellular activity was found in the homogenised yeast cells. 13-Hydroperoxy-(9Z,11E)-octadecadienoic acid (HPOD) was the best substrate followed by 9-HPOD and then 13-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13-HPOT) while no activity was observed with the 9-HPOT. The pH optima of rLeHPL were at pH 7 and 5, respectively, for the 13-HPOD and 13-HPOT substrates. GC/MS analyses confirmed that the major product was hexanal with a minor peak for nonanal.