融合表达过氧化氢酶提高多铜氧化酶稳定性及降解生物胺能力

Fusion expression with catalase improves the stability of multicopper oxidase and its efficiency in degrading biogenic amines

多铜氧化酶(Multicopper oxidase,MCO)家族中的某些酶可以通过氧化反应降解食品中的胺类危害物生物胺。然而酶在催化时因为持续被氧化可能会影响整个反应过程中MCO的活性及稳定性,使酶的催化效率和降解生物胺的能力下降。文中成功在大肠杆菌EscherichiacoliBL21(DE3)中构建并表达了来源于发酵乳杆菌Lactobacillus fermentum的多铜氧化酶(MCOF)与枯草芽孢杆菌过氧化氢酶(CAT)的融合酶。8种融合酶对H2O2的耐受性提高了51%–68%,融合酶CAT&MCOF在降解组胺的过程中稳定性比MCOF提高了17.3%。以2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid), ABTS)为底物时,CAT&MCOF对底物的亲和力(Km)提高了1.0倍,催化效率(kcat/Km)提高了1.7倍,摩尔比酶活提高了1.2倍,且在酸性(pH 2.5–4.5)和中高温(35–55℃)条件下的稳定性都有不同程度的提高。此外,CAT&MCOF对腐胺、尸胺和组胺的降解率分别为31.7%、36.0%和57.8%,分别比MCOF提高了132.5%,45.7%和38.9%。多铜氧化酶与过氧化氢酶融合表达提高酶催化稳定性和催化效率的策略将为通过酶的分子改造提高其应用特性提供参考。

Some enzymes belonging to the multicopper oxidase(MCO) family can degrade the hazardous biogenic amine(BA) present in food. However, the oxidation of MCO in the process of degrading BAs may reduce its activity and stability,resulting in decreased catalytic efficiency. In this work, an MCO from Lactobacillus fermentum(MCOF) was fused with a Bacillus subtilis catalase(CAT) using different strategies and the fusion enzymes were respectively expressed in Escherichia coli BL21(DE3). The tolerance of eight fused MCOFs to H2 O2 increased by 51%–68%, and the stability of CAT&MCOF increased by 17%, compared to the wild type MCOF. Using 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid(ABTS) as a substrate, the substrate affinity(K_(m)), the catalytic efficiency(kcat/Km) and the molar specific activity of CAT&MCOF increased by 1.0-fold, 1.7-fold and 1.2-fold than those of MCOF, respectively. The stability of CAT&MCOF under acidic conditions(pH 2.5–4.5) and moderate temperatures(35–55 ℃) also improved. Moreover, the degradation rates of putrescine, cadaverine and histamine catalyzed by CAT&MCOF reached 31.7%, 36.0% and 57.8%, respectively, which increased by 132.5%, 45.7% and 38.9% compared to that of MCOF. The improvement on the stability and catalytic efficiency of MCOF by fusion expression with CAT provides a good example for improving the applicability of enzymes through molecular modifications.