重组脂肪氧合酶分子改造

Molecular modification of a recombinant lipoxygenase

脂肪氧合酶（Lipoxygenase,EC1.13.11.12,LOX）是一种双加氧酶,它能够专一催化多元不饱和脂肪酸中的cis,cis-1,4-戊二烯结构,通过分子加氢形成具有共轭双键的氢过氧化物。LOX在食品、医药和化工等领域都有广泛的应用,市场需求巨大。但微生物来源的LOX往往具有热稳定性较差的缺点,这限制了LOX在工业加工中的应用,因此提高酶的热稳定性对提高酶的应用价值和降低工业成本具有重要意义。在脂肪氧合酶N端与C端结构域链接处的loop结构（L6）分别插入1～3个L6序列,构建突变体L6 Ⅰ、L6 Ⅱ、L6 Ⅲ。3种突变体均可以在大肠杆菌中正常分泌表达,突变体的二级结构和三级结构与野生酶没有明显差异。L6 Ⅰ、L6 Ⅱ、L6 Ⅲ 3个突变体在50℃下的热稳定性分别是野生酶的2.3倍、3.2倍和4倍,其中突变体L6 Ⅱ、L6 Ⅲ的最适反应温度比野生酶提高了5、10℃。突变体的比酶活分别降低为野生酶的28.7%、57.9%和47.6%,同时其疏水性比野生酶提高了约300%。

Lipoxygenase （EC1.13.11.12, LOX） catalyse oxidation of polyunsaturated fatty acids in lipids containing a cis, cis-l,4-pentadiene structure to form Z, E conjugated hydroperoxides. It has potential important applications in food, paint and medicine industry. However, the thermo-instability of LOX from microorganism limited its application in aforementioned fields. In this study, the L6 region （TQRGQG） was inserted to the C-terminus of Gly^206 by one, two and three times resulted in mutants L6 I, L6 II and L6 III, respectively. Compared with the wild-type LOX, all the mutants were expressed extracellularly and the secondary and tertiary structure of the mutants were similar. The half-life times of the three mutants （L6 I, L6 II and L6 III） under 50 ℃ increased by 1.3-fold, 2.2-fold and 3-fold, respectively. The optimal temperature of L6 II and L6 III enhanced by 5 ℃ and 10 ℃, but the catalytic activity of L6 I, L6 II and L6 III were decreased 71.3%, 42.1% and 52.4% in contrast to the wild-type LOX. The hydrophobicity of the three mutants were all enhanced by nearly 3-folder.