Abstract: In order to study the mechanism of action of lipoxygenase (LOX) in lipid oxidation and flavor formation during the storage and processing of pork, the gene encoding the catalytic domain of porcine muscle 12-lipoxygenase (12-LOXcd) was obtained by sequence analysis and PCR amplification. The amplified gene was inserted into an inducible expression vector and expressed in E. coli by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The recombinant protein was purified by consecutive Ni-NTA affinity chromatography and Superdex 200 gel filtration chromatography. Then the enzymatic properties of the purified 12-LOXcd were studied. The results showed that the recombinant vector pMBP-12- LOXcd successfully expressed porcine 12-LOXcd in the culture supernatant of E. coli. With linoleic acid as substrate, the purified enzyme had a specific activity of 2 826.7 U/mg, and the maximum enzymatic activity was observed at pH 6.0 and 30 ℃. The Km values obtained for linoleic acid (Km= 0.40 mmol/L), linolenic acid (Km=0.55 mmol/L) and arachidonic acid (Km= 4.15 mmol/L) revealed preferential use of linoleic acid as the substrate. Compared with soybean LOX, 12-LOXcd could keep active at a high concentration of NaCl (9%). 12-LOXcd exhibited a better thermal stability than soybean LOX, and it was fully deactivated at 60 ℃. Besides, 12-LOXcd showed better pH stability than soybean LOX, and it could retain part of its activity under alkaline conditions.

Key words: pig muscle, lipoxygenase, prokaryotic expression, purification, enzymatic properties