Abstract: Lipoxygenase (LOX) is a kind of oxidoreductase containing non-heme iron in the active center, which is used in food, medicine and chemical industry. In our study, we first constructed recombinant Escherichia coli BL21(DE3) to express PaLOX and BtLOX from Pseudomonas aeruginosa and Burkholderia thalandensis, respectively, and compared their enzymatic properties. Enzymatic analysis showed that the optimum reaction temperature of recombinant PaLOX and BtLOX was 25 ℃ and 35 ℃, respectively. Their optimum reaction pH was 7 and 7.5. Furthermore, the Kcat/Km of PaLOX was 16.7 times higher than BtLOX and showed potential for industrial application. To improve soluble expression of PaLOX, the IPTG inducer was replaced with lactose, which increased the activity of intracellular LOX enzyme by 1.53 times, and the expression level of soluble protein was 2.41 times higher. Next, the activity of LOX increased 1.85 times and the level of soluble expression increased 3.19 times by fusion expressed LOX with triggering factor. These results provided an important reference for the efficient production and application of LOX.
[1] ELLEN H, SUSAN K, ALENA L, et al.Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia[J]. Phytochemistry, 2008, 69(16): 2 774-2 780. [2] ANON. Evolution and expansion of the prokaryote-like lipoxygenase family in the brown alga Saccharina japonica[J]. Frontiers in Plant Science, 2017, 8: 2 018. [3] RODCASEY, HUGHES R K J F B. Recombinant lipoxygenases and oxylipin metabolism in relation to food quality[J]. Food Biotechnology, 2004, 18(2): 135-170. [4] CASEY R, WEST S I, HARDY D, et al.New frontiers in food enzymology: Recombinant lipoxygenases[J]. Trends in Food Science & Technology, 1999, 10(9): 297-302. [5] 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. [6] SANTANO E, PINTO M D C, PEDRO M. Chlorpromazine oxidation by hydroperoxidase activity of covalent immobilized lipoxygenase[J]. Biotechnology and Applied Biochemistry, 2002, 36(2): 95-100. [7] MAHALIK S, SHARMA A K, MUKHERJEE K J.Genome engineering for improved recombinant protein expression in Escherichia coli[J]. Microbial Cell Factories, 2014, 13(1): 177. [8] 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): 9 419-9 427. [9] KATERYNA G, SABINE S, DAGMAR H, et al.Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns[J]. Microbiology Open, 2019, 8(7): e775. [10] STEFAN H, TERTIUS C, PIETER S.Lipoxygenases: From isolation to application[J]. Comprehensive Reviews in Food Science and Food Safety, 2017, 16(1): 199-211. [11] RUSSELL R J, GERIKE U, DANSON M J, et al.Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium[J]. Structure, 1998, 6(6): 351-361. [12] CIARÓN Ó' FÁGÁIN. Enzyme stabilization—recent experimental progress[J]. Enzyme and Microbial Technology, 2003, 33(2): 137-149. [13] 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): 5 793-5 800. [14] AN J U, KIM B J, HONG S H, et al.Characterization of an omega-6 linoleate lipoxygenase from Burkholderia thailandensis and its application in the production of 13-hydroxyoctadecadienoic acid[J]. Applied Microbiology and Biotechnology, 2015, 99(13): 5 487-5 497. [15] CAO S, CHEN H, ZHANG C, et al.Heterologous expression and biochemical characterization of two lipoxygenases in oriental melon, Cucumis melo var. makuwa Makino[J]. Plos One, 2016, 11(4): e0153801. [16] KELLE S, ZELENA K, KRINGS U, et al.Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris[J]. Protein Expression and Puripication, 2014, 95: 233-239. [17] TASAKI Y, TOYAMA S, KURIBAYASHI T, et al.Molecular characterization of a lipoxygenase from the basidiomycete mushroom Pleurotus ostreatus[J]. Bioscience Biotechnology and Biochemistry, 2013, 77(1): 38-45. [18] HUI Q, BINGJIE X, YUJUN H, et al.Expression, purification, and characterization of a novel acidic Lipoxygenase from Myxococcus xanthus[J]. Protein Expres. Purif., 2017, 138: 13-17. [19] VIDAL-MAS J, BUSQUETS M, MANRESA A.Cloning and expression of a lipoxygenase from Pseudomonas aeruginosa 42A2[J]. Antonie Van Leeuwenhoek, 2005, 87(3): 245-251. [20] BALCHIN D, HAYER-HARTL M, HARTL F U. In vivo aspects of protein folding and quality control [J]. Science, 2016, 353(6 294): aac 4354. [21] KRAMER G, PATZELT H, RAUCH T, et al.Trigger factor peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia coli[J]. Journal of Biological Chemistry, 2004, 279(14): 14 165-14 170. [22] SHARMA B, CHUGH L K.Two isoforms of lipoxygenase from mature grains of pearl millet [Pennisetum glaucum (L.) R. Br.]: Purification and physico-chemico-kinetic characterization[J]. Journal of Food Science & Technology, 2017, 54(6): 1 577-1 584. [23] 王卫东, 杨万根, 付湘晋. 白鲢鱼肌肉脂肪氧合酶的分离纯化与鉴定[J]. 食品科学, 2010, 31(23): 164-166. [24] AKIKO S, HENRIK S L, KENJI M, et al.Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae[J]. Journal of Bioscience and Bioengineering, 2018, 126(4): 436-444. [25] EGMOND M R, BRUNORI M, FASELLA P M.The steady-state kinetics of the oxygenation of linoleic acid catalysed by soybean lipoxygenase[J]. European Journal of Biochemistry, 2010, 61(1): 93-100. [26] 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. [27] KUHN H, WALTHER M, KUBAN R J. Mammalian arachidonate 15-lipoxygenases-Structure, function,biological implications [J]. Prostaglamdins & Other Lipid Mediators, 2002, 68-69: 263-290. [28] 王广圣,徐智,刘松,陈坚,堵国成.重组脂肪氧合酶分子改造[J].食品科技,2014,39(9):2-7. [29] 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 & Biotechnology, 2014, 98(4): 1 663-1 669.
2020, Vol. 46 
