Cloning and expression ofbenzaldehydelyase from Pseudomonas fluorescens Biovar I in Pichia pastoris

Büyüksungur, Arda
Benzaldehyde lyase (BAL, EC from Pseudomonas fluorescens Biovar I, a thiamine pyrophosphate (ThDP) dependent enzyme, catalyzes the enzymatic kinetic resolution of racemates by C-C bond cleavage and concomitant C-C bond formation. In this study, benzaldehyde lyase gene from Pseudomonas fluorescens Biovar I was cloned into Pichia pastoris, with the aim of the extracellular production of the enzyme. For this purpose, firstly, PCR amplified bal gene was cloned into an integration vector pPICZalphaA. Thereafter the recombinant plasmid pPICZalphaA::bal was transformed into P.pastoris. Extracellular benzaldehyde lyase enzyme was expressed under the control of the strong AOX promoter and the secretion of the enzyme in the fermentation medium was achieved by means of S. cerevisiae alpha factor signal sequence. The recombinant cells were grown for 48-72 hours in solid medium then the cells inoculated in glycerol containing medium. After being separated by centrifugation cells were transferred into methanol containing production medium. In methanol containing medium cells were grown for 72 h. Starting from t=24 h methanol was added to medium as an inducer of AOX promoter and the carbon source in order to produce BAL in every 24 hour. SDS-PAGE analyses illustrated that extracellular benzaldehyde lyase enzyme produced by the recombinant P.pastoris strain had the size of 59 kDa, which is the size of benzaldehyde lyase monomer. FPLC analysis showed that concentration of the tetrameric form of benzaldehyde lyase enzyme, active form, was much less than the monomeric form of the enzyme indicating that the enzyme produced by recombinant P.pastoris mostly could not fold into multimeric form in the fermentation medium.


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Citation Formats
A. Büyüksungur, “Cloning and expression ofbenzaldehydelyase from Pseudomonas fluorescens Biovar I in Pichia pastoris,” M.S. - Master of Science, Middle East Technical University, 2006.