Ni Xiumei, Fang Fang
Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Dec 25;37(12):4382-4394. doi: 10.13345/j.cjb.200799.
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 H2O2 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 (Km), 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 °C) 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.
一些属于多铜氧化酶（MCO）家族的酶能够降解食品中存在的有害生物胺（BA）。然而，MCO在降解BA的过程中发生氧化，可能会降低其活性和稳定性，导致催化效率下降。在本研究中，采用不同策略将来自发酵乳杆菌的MCO（MCOF）与枯草芽孢杆菌过氧化氢酶（CAT）融合，并在大肠杆菌BL21(DE3)中分别表达融合酶。与野生型MCOF相比，8种融合MCOF对H2O2的耐受性提高了51%-68%，CAT&MCOF的稳定性提高了17%。以2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）为底物时，CAT&MCOF的底物亲和力（Km）、催化效率（kcat/Km）和摩尔比活性分别比MCOF提高了1.0倍、1.7倍和1.2倍。CAT&MCOF在酸性条件（pH 2.5-4.5）和中等温度（35-55°C）下的稳定性也有所提高。此外，CAT&MCOF催化腐胺、尸胺和组胺的降解率分别达到31.7%、36.0%和57.8%，与MCOF相比分别提高了132.5%、45.7%和38.9%。通过与CAT融合表达提高MCOF的稳定性和催化效率，为通过分子修饰提高酶的适用性提供了一个很好的例子。
