Abstract:

The phenylalanine ammonia lyase gene (PAL) was cloned from Olea europaea by homology cloning, RT-PCR,
FPNI-PCR (fusion primer and nested integrated PCR) and 3’-RACE, and named as OePAL. Sequencing showed that the
full-length DNA of OePAL was 2 970 bp (GenBank, KJ511867) with an intron (393–1 220 bp). Meanwhile, the full-length
cDNA of OePAL was 2 142 bp (GenBank, KJ511868). The open reading frame (ORF) of OePAL encoded 713 amino acid
residues, and sequence analysis suggested that OePAL had high homology with other botanic PALs. The full-length exon
of OePAL expression vector pPICZα A-OePAL was constructed and expressed in Pichia pastoris strain X33. SDS-PAGE
analysis showed that the molecular weight of recombinant enzyme was approximately 77.5 kD, and the specific activity
of purified enzyme was 196.3 U/mg. The enzymatic properties of recombinant 6×His-OePAL indicated that the optimum
reaction conditions were 40 ℃ and pH 8.8. Ions such as Cu2+ and Na+ could enhance the enzyme activity within the tested
range. The enzyme had a Km of 4.89 × 10-4 mol/L for L-phenylalanine at the optimum reaction conditions. Results indicated
that OePAL was amplified and sub-cloned into the vector of pPICZα A, and its function was validated successfully.

Key words: phenylalanine ammonia lyase (PAL), gene cloning, expression vector, Pichia pastoris strain X33