猪肉12-脂肪氧合酶催化结构域的表达、纯化及其酶学性质研究

摘要/Abstract

摘要： 为研究脂肪氧合酶（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，本研究通过序列分析和PCR扩增获得了猪12-脂肪氧合酶的催化结构域（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是2826.7 U/mg，最适pH为6.0，最适作用温度为30 ℃。与大豆LOX相比，该酶在较高NaCl浓度（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稳定性也优于大豆LOX，在碱性条件下仍能保留部分活力。

关键词: 猪肉, 脂肪氧合酶, 表达, 纯化, 酶学性质

Abstract: In order to study the mechanism of lipoxygenase (LOX) during the storage and processing of pork, the catalytic domain of porcine 12-lipoxygenase catalytic domain (12-LOXcd) was obtained by sequence analysis and PCR amplification. The coding sequence of 12-LOXcd was constructed into an inducible expression vector and expressed in E. coli induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).The recombinant protein was purified by Ni-NTA affinity chromatography and Superdex 200 gel filtration chromatography. Then enzymatic properties of the purified 12-LOXcd were studied. The results showed that the recombinant vector pMBP-12-LOXcd successfully expressed pig 12-LOXcd in the supernatant of E. coli. With linoleic acid as substrate, the purified enzyme had a specific activity of 2826.7 U/mg, a maximum activity at pH 6.0, and the optimal temperature of 30 °C. Compared with soybean LOX, 12-LOXcd could keep active at high concentration of NaCl (9%). 12-LOXcd exhibited a better thermal stability than soybean LOX, and it was fully deactivated at 60 °C. Besides, 12-LOXcd showed better pH stability than soybean LOX, and it could retain part of its activity under alkaline conditions.

Key words: Pig, lipoxygenase, prokaryotic expression, purification, enzymatic properties

中图分类号:

Q816

李鹏鹏. 猪肉12-脂肪氧合酶催化结构域的表达、纯化及其酶学性质研究[J]. 食品科学, 0, (): 0-0.

参考文献

[1] 闫静芳, 王红霞, 郭玉鑫, 等.脂肪氧合酶的研究及应用进展[J]. 食品安全质量检测学报, 2013(3):799-805.

[2] Alberti J C, Mariani M, Gambotti C, et al.A functional role identified for conserved charged residues at the active site entrance of lipoxygenase with double specificity[J]. Journal of Molecular Catalysis B Enzymatic, 2016, 123:167-173. DOI: 10.1016/j.molcatb.2015.10.011.

[3]Liavonchanka A, Feussner I.Lipoxygenases: occurrence,functions and catalysis[J].Journal of Plant Physiology, 2006, 163(3):348-357.

[4] 曲清莉, 傅茂润, 代红飞.脂氧合酶(LOX)在脂肪酸氧化中的作用研究进展[J].食品研究与开发, 2015(10):137-142. DOI:10.3969/j.issn.1005-6521.2015.10.037.

[5]刘松, 陆信曜, 周景文, 等.脂肪氧合酶结构、分子改造与发酵研究进展[J].生物技术通报, 2015, 31(12):34-41.
[6]Rankin S M, Parthasarathy S, Steinberg D.Evidence for a dominant role of lipoxygenase(s) in the oxidation of LDL by mouse peritoneal macrophages[J].Journal of Lipid Research,1991,32(3):449.

[7] Tayeb A H, Sadeghifar H, Hubbe M A, et al.Lipoxygenase-mediated peroxidation of model plant extractives[J]. Industrial Crops & Products, 2017, 104:253-262. DOI: 10.1016/j.indcrop.2017.04.041.
[8] Andre E, Hou KW.The presence of a lipid oxidase in soybean. [J]. Lieb CR Acad Sci, 1932, 194: 645–647.

[9]Fu X, Xu S, Wang Z.Kinetics of lipid oxidation and off-odor formation in silver carp mince: The effect of lipoxygenase and hemoglobin[J].Food Research International, 2009, 42(1):85-90.

[10]Fu X, Lin Q, Xu S, et al.Effect of drying methods and antioxidants on the flavor and lipid oxidation of silver carp slices[J].LWT - Food Science and Technology, 2015, 61(1):251-257.

[11]汪晓鸣, 陆兆新.脂肪氧合酶在农产食品中应用的研究进展[J].核农学报, 2013, 27(10):1547-1552.

[12]Qiuxing, Yanyan, Laihao, et al.Lipid Oxidation and Fatty Acid Composition in Salt-Dried YellowCroaker(Pseudosciaena polyactis) During Processing[J].中国海洋大学学报英文版, 2017, 16(5):855-862.

[13] 郇延军.金华火腿加工过程中脂类物质及风味成分变化的研究[D]. 南京农业大学, 2005.

[14]Roldan M, Antequera T, Armenteros M, et al.Effect of different temperature–time combinations on lipid and protein oxidation of sous-vide cooked lamb loins[J].Food Chemistry, 2014, 149(149):129-136.

[15] 曹锦轩, 吕彤, 王颖, 等.脂肪相关酶类在干腌肉制品风味形成过程中的作用[J].现代食品科技, 2015(1):254-258.DOI: 10.13982/j.mfst.1673-9078.2015.1.043.

[16]吴宝森, 孙玥晖, 刘姝韵, 等.肉和肉制品中脂质氧化的研究进展[J].食品安全质量检测学报, 2017, 8(3):814-818.

[17]Navicha W B, Hua Y, Masamba K, et al.Optimization of soybean roasting parameters in developing nutritious and lipoxygenase free soymilk[J].Journal of Food Measurement & Characterization, 2017, 11(4):1899-1908.

[18] Stephany M, Bader-Mittermaier S, Schweiggert-Weisz U, et al.Lipoxygenase activity in different species of sweet lupin (Lupinus L.) seeds and flakes[J]. Food Chemistry, 2015, 174:400. DOI: 10.1016/j.foodchem.2014.11.029.

[19]Mandal S, Dahuja A, Santha I M.Lipoxygenase activity in soybean is modulated by enzyme-substrate ratio[J].Journal of Plant Biochemistry & Biotechnology, 2014, 23(2):217-220.

[20] Mashima R, Okuyama T.The role of lipoxygenases in pathophysiology; new insights and future perspectives[J]. Redox Biology, 2015, 6:297-310. DOI: 10.1016/j.redox.2015.08.006.

[21]张迎阳.干腌肉制品中脂质自动氧化机理及调控机制研究[D]. 南京农业大学, 2014.

[22]Gata J L, And M C P, Macías P.Lipoxygenase Activity in Pig Muscle:Purification and Partial Characterization[J].Journal of Agricultural & Food Chemistry, 1996, 44(9):2573-2577.

[23]Jin G, Zhang J, Yu X, et al.Crude lipoxygenase from pig muscle: partial characterization and interactions of temperature,NaCl and pH on its activity[J].Meat Science, 2011, 87(3):257-263
[24]张充, 周孝伟, 吕凤霞, 等.重组鱼腥藻脂肪氧合酶基因的克隆表达、分离纯化及活性分析[J].生物工程学报, 2012, 28(4):440-456.

[25]何立超, 马素敏, 李成梁, 等.温度、盐分以及值对鸭肉脂肪氧合酶活性的交互影响[J].江苏农业学报, 2016, 32(6):1404-1409.

[26]Kermasha S, Metche M.Characterization of seed lipoxygenase of Phaseolus vulgaris cv,Haricot[J].Journal of Food Science, 1986, 51(5):1224-1227.

[27]Szymanowska U, Jakubczyk A, Baraniak B, et al.Characterisation of lipoxygenase from pea seeds ( Pisum sativum,varTelephone,L.)[J].Food Chemistry, 2009, 116(4):906-910.

[28] Bataille L, 李亚茹.以麦芽糖结合蛋白作水溶性标签的疏水类弹性蛋白多肽的表达与纯化[J].中国医药工业杂志, 2016(2):245-245.

[29]陈欣, 姚忠, 徐为民, 等.麻鸭脂肪氧合酶的分离纯化及其性质研究[J].食品与发酵工业, 2013, 39(12):39-43.

[30]Sudharshan E, Srinivasulu S, Rao A G A.pH-induced domain interaction and conformational transitions of lipoxygenase-1[J].Biochimica Et Biophysica Acta, 2000, 1480(1-2):13.DOI: 10.1016/S0167-4838(00)00099-6.