细菌脂肪氧合酶酶学性质分析及表达优化

庞翠萍; 刘松; 周景文; 张国强; 李江华

doi:10.13995/j.cnki.11-1802/ts.024233

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食品与发酵工业 ›› 2020, Vol. 46 ›› Issue (19) : 1-8. DOI: 10.13995/j.cnki.11-1802/ts.024233

研究报告

细菌脂肪氧合酶酶学性质分析及表达优化

庞翠萍^1,2, 刘松^1,2, 周景文^1,2, 张国强^1,2,*, 李江华^2,*

作者信息 +

Characterization and expression optimization of lipoxygenase from bacteria

PANG Cuiping^1,2, LIU Song^1,2, ZHOU Jingwen^1,2, ZHANG Guoqiang^1,2,*, LI Jianghua^2,*

Author information +

文章历史 +

摘要

脂肪氧合酶(lipoxygenase,LOX)是一类含有非血红素铁的双加氧酶,在食品、医药和化工等行业具有广泛应用。利用Escherichia coli BL21(DE3)重组表达来源于Pseudomonas aeruginosa和Burkholderia thailandensis的脂肪氧合酶PaLOX、BtLOX,并对其酶学性质进行比较分析。通过对酶学性质分析发现:PaLOX、BtLOX的最适反应温度分别为25和35 ℃;最适反应pH分别为7.0和7.5。PaLOX的K_cat/K_m较BtLOX高16.7倍,展现出更好的开发应用潜力。为优化PaLOX的可溶性表达,采取了2种优化策略:首先利用乳糖作为诱导剂代替异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG),使胞内酶活力提高了1.53倍,可溶表达水平提高了2.41倍;进一步与触发因子(triggering factor, TF)融合表达,TF-PaLOX酶活力提高了1.85倍,可溶表达水平提高3.19倍。该研究通过分析细菌来源脂肪氧合酶酶学性质及优化表达条件,为脂肪氧合酶的高效生产及应用提供了重要参考。

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 K_cat/K_m 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.

导出引用

庞翠萍, 刘松, 周景文, 张国强, 李江华. 细菌脂肪氧合酶酶学性质分析及表达优化[J]. 食品与发酵工业, 2020, 46(19): 1-8 https://doi.org/10.13995/j.cnki.11-1802/ts.024233

PANG Cuiping, LIU Song, ZHOU Jingwen, ZHANG Guoqiang, LI Jianghua. Characterization and expression optimization of lipoxygenase from bacteria[J]. Food and Fermentation Industries, 2020, 46(19): 1-8 https://doi.org/10.13995/j.cnki.11-1802/ts.024233

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