Benzaldehyde lyase (BAL, EC 4.1.2.38) 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.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation and salt, probably by stabilizing protein structures and lipid membranes. Trehalose-6-phosphate synthase 1 (TPS1) is a subunit of trehalose synthase complex in fungi / it plays a key role in the biosynthesis of trehalose. In this study, a TPS1 gene fragment in R. oryzae was cloned successfully by PCR with primers designed according to eight hypothetical proteins found from BLAST search which was performed by using S. cerevisiae TPS1 gene template. The full length of R. oryzae TPS1 gene (designated RoTPS1) was attained by RTPCR with primers specific to the 3&amp / #8242 / and 5&amp / #8242 / end of the RoTPS1 cDNA. The RoTPS1cDNA was composed of 2505 bps encoding a protein of 834 amino acids with a molecular mass of 93.8 kDa. The amino acid sequence has relatively high homology with the TPS1s of several other organisms. RoTPS1 was further characterized by transformation into S. cerevisiae tps1 mutant. In galactose media, the growth curves of wild type, tps1 mutant and transformant S. cerevisiae cells had a comparable pattern in general, tps1 mutant reached to a higher maximum cell concentration compared to the others and wild type had a slightly lower specific growth rate compared to the tps1 mutant and transformed cells. Trehalose levels of transformant and wild type cells were increased up to 37 mg/gdw in the stationary phase.