植物乳杆菌源胺氧化酶的异源表达及功能结构分析

Heterologous expression, function and structure of amine oxidase derived from Lactiplantibacillus plantarum

摘要:

【目的】生物胺是一类广泛存在发酵食品中潜在有害物质，可被胺氧化酶分解。本研究对来源于乳酸菌的胺氧化酶的酶学性质及其降生物胺能力进行了探究。【方法】本研究在大肠杆菌中异源表达了植物乳杆菌中多铜氧化酶基因SufI，经过优化表达条件与纯化重组酶后，分析了该酶的最适反应条件、酶稳定性、降生物胺能力、光谱与结构特性。【结果】重组酶的最适pH值为3.5，最适温度为20 ℃，在pH 4.0-10.0或15-65 ℃的条件下，相对酶活力保持在70%以上。重组酶具有较好的稳定性，酶活不受乙醇等抑制剂的影响。在8种生物胺的混合体系内，重组酶对生物胺总量的降解量可达403.23 μg/mL，其中对酪胺的降解量最多，超过70 μg/mL (34.99%)。在单一生物胺体系内，重组酶对酪的底物亲和性较高，酶活可达18.33 U/mL。紫外-可见扫描光谱显示酶蛋白在600 nm处有多铜氧化酶家族特征吸收峰，傅里叶红外光谱解析酰胺Ⅰ带中α螺旋、β折叠、β转角和无规则卷曲的相对含量分别为21.52%、20.72%、33.80%和23.97%。同源建模预测该酶具有3个铜结合域，且含有组氨酸、半胱氨酸、甲硫氨酸和谷氨酸等铜配体结合的氨基酸残基位点，结果表明重组酶属于多铜氧化酶家族。【结论】植物乳杆菌源多铜氧化酶具有较好的酸碱稳定性和热稳定性，能够降解酪胺等多种生物胺，有望应用于发酵食品及饮料中生物胺的控制。

Abstract:

[Objective] Biogenic amines (BAs) widely exist in fermented food as a group of potentially harmful substances which can be degraded by amine oxidases. This study aims to explore, the enzymatic properties and amine-degrading abilities of amine oxidases from lactic acid bacteria. [Methods] The recombinant protein of the multicopper oxidase gene SufI in Lactobacillus plantarum was obtained through heterologous expression. After expression condition optimization and purification, the optimal reaction conditions, enzyme stability, amine-reducing ability, spectrum, and structural characteristics of the recombinant protein were investigated. [Results] The recombinant enzymes had the optimal performance at pH 3.5 and 20 ℃. The relative activity of the recombinant enzyme was above 70% within pH 4.0–10.0 and 15–65 ℃. This enzyme showed good stability, and was not affected by inhibitors such as ethanol. In a reaction system containing eight BAs, the recombinant enzyme could degrade 403.23 μg/mL and showed the highest degradation rate of 34.99% (70 μg/mL) for tyramine. In a reaction system containing single BAs, the recombinant enzyme had highest substrate specificity affinity toward tyramine, demonstrating the activity of 18.33 U/mL. The UV-visible scanning spectrum showed that the enzyme had a characteristic absorption peak at 600 nm. According to Fourier transform infrared spectrum, the relative content of alpha helixes, beta sheets, beta turns, and random coils in the secondary structure of amide was 21.52%, 20.72%, 33.80%, and 23.97%, respectively. Homologous modeling predicted that the enzyme possessed three copper-binding domains, which contained histidine, cysteine, methionine, glutamate, and other residues. The structure indicated that the recombinant enzyme belonged to the multicopper oxidase family. [Conclusion] The multicopper oxidase from L. plantarum JB1 showed good aciD-base and thermal stability, and was capable of degrading BAs, especially tyramine. It is expected to be further developed for fermented food and beverages.