Cloning of allanase gene from garlic bulb and its expression in Pichia pastoris / 中草药

ABSTRACT

Objective To clone the alliinase gene from the garlic bulb and construct the eukaryote expression plasmid for expressing the recombinant alliinase in Pichia pastoris system and analyzing its bioactivity. Metheds The alliinase gene was cloned from the Zhejiang garlic bulb by RT-PCR and the eukaryote expression plasmid of alliinase was constructed with the pPIczαC vector. The recombinant plasmid was transformed into Pichia pastoris X-33 by eletroporation. The positive clones were screened and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and Western blotting. The activities of the recombinant protein and the extracted alliinase were detected by the pyruvic acid method and compared by specific activity. The contents of the two kinds of alliinase were detected by Lowry method. Results The alliinase gene was successfully cloned from the garlic bulb, the length of alliinase gene was 1 500 bp, the molecule of the recombinant alliinase was about 5.5 × 104, existed in the supernatant of Pichia pastoris. The specific activity of the recombinant protein was (82.09 ± 3.89) U/mg and the nature alliinase was (176.49 ± 5.06) U/mg. Conlusion The alliinase gene is successfully expressed in Pichia pastoris system. The recombinant alliinase has the activity of enzyme, but is lower than that of the extracted alliinase.