脂肪氧合酶结构、分子改造与发酵研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.005

摘要/Abstract

摘要： 脂肪氧合酶(EC 1.13.11.12)能专一催化氧化含有Z，Z-1，4-戊二烯结构的多元不饱和脂肪酸，形成具有共轭双键的脂肪酸氢过氧化物，广泛应用于食品加工、化工、医药等领域。1932年首次在大豆中发现脂肪氧合酶以来，人们已在多种动植物组织和微生物中检测到脂肪氧合酶。广泛的来源使脂肪氧合酶结构亦呈现多样性，包括经典结构、融合结构、无N端Β-折叠结构及含锰离子结构等。为提高应用性能，定点突变和融合自组装双亲短肽等分子改造技术已用于提高脂肪氧合酶比酶活和热稳定性。基于发酵法的天然优势，构建高产脂肪氧合酶的重组菌成为近年研究的热点。总结了典型脂肪氧合酶的结构、分子改造及发酵法生产研究进展，旨为后续研究提供参考。

关键词: 脂肪氧合酶, 结构与功能, 分子改造, 发酵

Abstract: Lipoxygenases(EC1.13.11.12)catalyze and oxidize the polyunsaturated fatty acids containing Z, Z-1, 4-pentadiene structures to form the conjugated hydroperoxides of fatty acid, and they are widely used in food industry, chemical industry and pharmaceutical industry. Since lipoxygenase was firstly discovered in soybean in 1932, it has been detected in many animal and vegetable tissues as well as microorganisms. Broad sources led to various structure types of lipoxygenase, including classic, fusing, Β-sheet deleted, and Mn²⁺-containing structures. To improve the application performance, molecular modification techniques, such as site-directed mutagenesis and fusing with self-assembling amphiphilic peptides, have been used to enhance the specific activity and thermal stability of lipoxygenase. Considering the natural advantages of fermentation method, the construction of recombinant strains producing high-yield lipoxygenase has become the research hotspot recently. The advances on the structure, molecular modification, and fermentation of classic lipoxygenases were briefly summarized for providing a reference for further studies.

Key words: lipoxygenase, structure and function, molecular modification, fermentation

刘松,陆信曜,周景文,堵国成,陈坚. 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.

Liu Song, Lu Xinyao, Zhou Jingwen, Du Guocheng, Chen Jian. Research Advance on the Structure, Molecular Modification, and Fermentation of Lipoxygenases[J]. Biotechnology Bulletin, 2015, 31(12): 34-41.

参考文献

[1]Nyyssola A, Heshof R, Haarmann T, et al. Methods for identifying lipoxygenase producing microorganisms on agar plates[J]. AMB Express, 2012, 2:17.
[2]Coffa G, Brash AR. A single active site residue directs oxygenation stereospecificity in lipoxygenases:stereocontrol is linked to the position of oxygenation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(44):15579-15584.
[3]Liavonchanka A, Feussner I. Lipoxygenases:occurrence, functions and catalysis[J]. Journal of Plant Physiology, 2006, 163(3):348-357.
[4]Andre E, Hou KW. The presence of a lipid oxidase in soybean, Glycine soya[J]. Compte Rendu Acad Sci(Paris), 1932, 194:645-647.
[5] Howe GA, Jander G. Plant immunity to insect herbivores[J]. Annual Review of Plant Biology, 2008, 59:41-66.
[6] Mosblech A, Feussner I, Heilmann I. Oxylipins:structurally diverse metabolites from fatty acid oxidation[J]. Plant Physiol Biochem, 2009, 47(6):511-517.
[7] Yu Z, Crichton I, Tang SY, et al. Disruption of the 5-lipoxygenase pathway attenuates atherogenesis consequent to COX-2 deletion in mice[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(17):6727-6732.
[8] Aggarwal NT, Gauthier KM, Campbell WB. Endothelial nitric oxide and 15-lipoxygenase-1 metabolites independently mediate relaxation of the rabbit aorta[J]. Vascul Pharmacol, 2012, 56(1-2):106-112.
[9] Skrzypczak-Jankun E, Jankun J, Al-Senaidy A. Human lipoxygenase:developments in its structure, function, relevance to diseases and challenges in drug development[J]. Current Medicinal Chemistry, 2012, 19(30):5122-5127.
[10]Hersberger M. Potential role of the lipoxygenase derived lipid mediators in atherosclerosis:leukotrienes, lipoxins and resolvins[J]. Clinical Chemistry and Laboratory Medicine, 2010, 48(8):1063-1073.
[11]Wymann MP, Schneiter R. Lipid signalling in disease[J]. Nature Reviews Molecular Cell Biology, 2008, 9(2):162-176.
[12]Weinberger F, Lion U, Delage L, et al. Up-regulation of lipoxygenase, phospholipase, and oxylipin-production in the induced chemical defense of the red alga Gracilaria chilensis against epiphytes[J]. Journal of Chemical Ecology, 2011, 37(7):677-686.
[13]Brodhun F, Feussner I. Oxylipins in fungi[J]. FEBS Journal, 2011, 278(7):1047-1063.
[14]Shechter G, Grossman S. Lipoxygenase from baker's yeast:purification and properties[J]. International Journal of Biochemistry, 1983, 15(11):1295-1304.
[15]Joo YC, Oh DK. Lipoxygenases:Potential starting biocatalysts for the synthesis of signaling compounds[J]. Biotechnology Advances, 2012, 30(6):1524-1532.
[16]Hughes DT, Sperandio V. Inter-kingdom signalling:communication between bacteria and their hosts[J]. Nature Reviews Microbio-logy, 2008, 6(2):111-120.
[17]Koeduka T, Kajiwara T, Matsui K. Cloning of lipoxygenase genes from a cyanobacterium, Nostoc punctiforme, and its expression in Eschelichia coli[J]. Current Microbiology, 2007, 54(4):315-319.
[18]Lang I, Gobel C, Porzel A, et al. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120[J]. Biochemical Journal, 2008, 410(2):347-357.
[19]Vance R, Hong S, Gronert K, et al. The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(7):2135.
[20]Ivanov I, Heydeck D, Hofheinz K, et al. Molecular enzymology of lipoxygenases[J]. Archives of Biochemistry and Biophysics, 2010, 503(2):161-174.
[21]Casey R, West SI, Hardy D, et al. New frontiers in food enzymology:recombinant lipoxygenases[J]. Trends In Food Science & Technology, 1999, 10(9):297-302.
[22]Buchhaupt M, Guder JC, Etschmann MM, et al. Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase[J]. Applied Microbiology and Biotechnology, 2012, 93(1):159-168.
[23]Noverr MC, Toews GB, Huffnagle GB. Production of prostaglandins and leukotrienes by pathogenic fungi[J]. Infection and Immunity, 2002, 70(1):400-402.
[24] 林影. 生物酶在造纸工业绿色制造中的应用[J]. 生物工程学报, 2014, 30(1):83-89.
[25]Casey R, Hughes RK. Recombinant lipoxygenases and oxylipin metabolism in relation to food quality[J]. Food Biotechnology, 2004, 18(2):135-170.
[26]Schneider C, Niisuke K, Boeglin WE, et al. Enzymatic synthesis of a bicyclobutane fatty acid by a hemoprotein lipoxygenase fusion protein from the cyanobacterium Anabaena PCC 7120[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(48):18941-18945.
[27]Cristea M, Oliw EH. A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity:mechanism of biosynthesis of epoxyalcohols[J]. Journal of Biological Chemistry, 2006, 281(26):17612-17623.
[28]Minor W, Steczko J, Boguslaw Stec O, et al. Crystal structure of soybean lipoxygenase L-1 at 1. 4 ? resolution[J]. Biochemistry, 1996, 35(33):10687-10701.
[29]Gilbert NC, Niebuhr M, Tsuruta H, et al. A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex[J]. Biochemistry, 2008, 47(40):10665-10676.
[30] Garreta A, Carpenai Vilella X, Busquets Abió M, et al. Crystalliza-tion and resolution of the lipoxygenase of Pseudomonas aeruginosa 42A2 and phylogenetic study of the subfamilies of the lipoxygenases[M]// Mu?oz-Torrero D. Recent Advances in Pharmaceutical Sci-ences, India:Transworld Research Network, 2011:247-273.
[31]Corbin JA, Evans JH, Landgraf KE, et al. Mechanism of specific membrane targeting by C2 domains:localized pools of target lipids enhance Ca²⁺ affinity[J]. Biochemistry, 2007, 46(14):4322-4336.
[32]Chahinian H, Sias B, Carriere F. The C-terminal domain of pancreatic lipase:functional and structural analogies with C2 domains[J]. Current Protein and Peptide Science, 2000, 1(1):91-103.
[33]Dainese E, Angelucci CB, Sabatucci A, et al. A novel role for iron in modulating the activity and membrane-binding ability of a trimmed soybean lipoxygenase-1[J]. FASEB Journal, 2010, 24(6):1725-1736.
[34]Walther M, Anton M, Wiedmann M, et al. The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding[J]. Journal of Biological Chemistry, 2002, 277(30):27360-27366.
[35]Oldham ML, Brash AR, Newcomer ME. Insights from the X-ray crystal structure of coral 8R-lipoxygenase:calcium activation via a C2-like domain and a structural basis of product chirality[J]. Journal of Biological Chemistry, 2005, 280(47):39545-39552.
[36]Kulkarni S, Das S, Funk CD, et al. Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase[J]. Journal of Biological Chemistry, 2002, 277(15):13167.
[37]Hammel M, Walther M, Prassl R, et al. Structural flexibility of the N-terminal beta-barrel domain of 15-lipoxygenase-1 probed by small angle X-ray scattering. Functional consequences for activity regulation and membrane binding[J]. Journal of Molecular Biology, 2004, 343(4):917-929.
[38]Eek P, J?rving R, J?rving I, et al. Structure of a calcium-dependent 11R-Lipoxygenase suggests a mechanism for Ca²⁺ regulation[J]. Journal of Biological Chemistry, 2012, 287(26):22377-22386.
[39]Saam J, Ivanov I, Walther M, et al. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels[J]. Proceedings of the National Academy of Sciences of of the United States Of America, 2007, 104(33):13319.
[40]Lu X, Zhang J, Liu S, et al. Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli[J]. Applied Microbiology and Biotechnology, 2013, 97(13):5793-5800.
[41]Hornung E, Walther M, Kuhn H, et al. Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(7):4192-4197.
[42]Sloane DL, Leung R, Craik CS, et al. A primary determinant for lipoxygenase positional specificity[J]. Nature, 1991, 354(6349):149-152.
[43]Gillmor SA, Villasenor A, Fletterick R, et al. The structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity[J]. Nature Structural Biology, 1997, 4(12):1003-1009.
[44]Schneider C, Pratt DA, Porter NA, et al. Control of oxygenation in lipoxygenase and cyclooxygenase catalysis[J]. Chemistry & biology, 2007, 14(5):473-488.
[45]Coffa G, Brash A. A single active site residue directs oxygenation stereospecificity in lipoxygenases:stereocontrol is linked to the position of oxygenation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(44):15579.
[46]Coffa G, Schneider C, Brash AR. A comprehensive model of positional and stereo control in lipoxygenases[J]. Biochemical and Biophysical Research Communications, 2005, 338(1):87-92.
[47]Kobe MJ, Neau DB, Mitchell CE, et al. The structure of human 15-Lipoxygenase-2 with a substrate mimic[J]. Journal of Biological Chemistry, 2014, 289(12):8562-8569.
[48]Neau DB, Bender G, Boeglin WE, et al. Crystal structure of a lipoxygenase in complex with substrate the arachidonic acid-binding site of 8R-lipoxygenase[J]. Journal of Biological Chemistry, 2014, 289(46):31905-31913.
[49]Maccarrone M, Salucci ML, van Zadelhoff G, et al. Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability[J]. Biochemistry, 2001, 40(23):6819-6827.
[50]Di Venere A, Salucci ML, van Zadelhoff G, et al. Structure-to-function relationship of mini-lipoxygenase, a 60-kDa fragment of soybean lipoxygenase-1 with lower stability but higher enzymatic activity[J]. Journal of Biological Chemistry, 2003, 278(20):18281-18288.
[51]Palmieri-Thiers C, Alberti JC, Canaan S, et al. Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies[J]. Archives of Biochemistry and Biophysics, 2011, 509(1):82-89.
[52]Lu X, Liu S, Feng Y, et al. Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions[J]. Applied Microbiology and Biotechnology, 2013, 97(21):9419-9427.
[53]Lu X, Liu S, Zhang D, et al. Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides[J]. Applied Microbiology and Biotechnology, 2013, 97(21):9419-9427.
[54]Herman RP, Hamberg M. Properties of the soluble arachidonic acid 15-lipoxygenase and 15-hydroperoxide isomerase from the oomycete Saprolegnia parasitica[J]. Prostaglandins, 1987, 34(1):129-139.
[55]Cristea M, Engstrom K, Su C, et al. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands[J]. Archives of Biochemistry and Biophysics, 2005, 434(1):201-211.
[56]Bae JH, Hou CT, Kim HR. Thermostable lipoxygenase is a key enzyme in the conversion of linoleic acid to trihydroxy-octadecenoic acid by Pseudomonas aeruginosa PR3[J]. Biotechnology and Bioprocess Engineering, 2010, 15(6):1022-1030.
[57]Steczko J, Donoho GA, Dixon JE, et al. Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli[J]. Protein Expression and Purification, 1991, 2(2-3):221-227.
[58]Knust B, Wettstein D. Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 1992, 37(3):342-351.
[59] Reddy RG, Yoshimoto T, Yamamoto S, et al. Expression, purifica-tion, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris[J]. Biochemical and Biophysical Research Communications, 1994, 205(1):381-388.
[60]Vidal-Mas J, Busquets M, Manresa A. Cloning and expression of a lipoxygenase from Pseudomonas aeruginosa 42A2[J]. Antonie Van Leeuwenhoek International Journal, 2005, 87(3):245-251.
[61]Zhang C, Tao T, Ying Q, et al. Extracellular production of lipoxygenase from Anabaena sp. PCC 7120 in Bacillus subtilis and its effect on wheat protein[J]. Applied Microbiology and Biotechnology, 2012, 94(4):949-958.
[62]陆信曜, 徐智, 刘松, 等. 重组大肠杆菌生产脂肪氧合酶的发酵优化[J]. 食品科技, 2013, 5:31-36.