猪肉12-脂肪氧合酶催化结构域的表达、纯化及其酶学性质分析

研究脂肪氧合酶（lipoxygenase，LOX）在猪肉贮藏、加工过程中对脂肪氧化及风味形成的作用机制。通过序列分析和聚合酶链式反应扩增获得了猪肉12-脂肪氧合酶催化结构域（12-lipoxygenase catalytic domain，12-LOXcd）的编码基因，采用大肠杆菌表达系统，经镍柱亲和层析和Superdex G200凝胶过滤层析纯化得到12-LOXcd蛋白，并研究其酶学性质。结果表明，构建的原核表达载体pMBP-12-LOXcd在大肠杆菌中成功可溶性表达了猪肉12-LOXcd，该重组蛋白经纯化可达电泳纯。12-LOXcd以亚油酸为底物的比活力为2 826.7 U/mg，最适pH值为6.0，最适作用温度为30 ℃。亚油酸Km为0.40 mmol/L，亚麻酸Km为0.55 mmol/L，花生四烯酸Km为4.15 mmol/L，表明最适底物为亚油酸。与大豆LOX相比，该酶在较高NaCl质量分数（9%）时仍保持活性稳定；对热较不稳定，在60 ℃条件下失活，但优于大豆LOX的热稳定性；此外，12-LOXcd的pH值稳定性也优于大豆LOX，在碱性条件下仍能保留部分活力。

Expression,Purification and Characterization of 12-Lipoxygenase Catalytic Domain from Pig Muscle

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.