枯草芽孢杆菌WTX1胞外酶降解AFB1酶学特性及降解位点分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1384

摘要/Abstract

摘要：

为丰富生物降解黄曲霉毒素B₁（AFB₁）资源，研究已筛选到的枯草芽孢杆菌WTX1降解AFB₁的胞外酶特性及其降解位点。通过降解与AFB₁毒性位点结构相似的3种化合物，采用液相色谱串联三重四级杆质谱（UPLC-MS/MS）检测降解液。经层析分离，降解AFB₁胞外酶主要有Ea、Eb两种。最适降解条件：降解酶Ea 降解温度为50℃，pH 5，降解率高达88.4%；降解酶Eb降解温度为37℃，pH 5，降解率为73.4%；Ea酶的作用位点为AFB₁的呋喃环双键、香兰素内脂环、戊烯酮环结构，Eb酶的作用位点为AFB₁的香兰素内脂环、戊烯酮环结构。酶组降解途径为通过水解作用，加氢及连续脱去-CO基团。菌株WTX1胞外酶通过水解作用破坏AFB₁毒性位点，起到降解作用，该酶具有很好的抗逆性，温度和pH适应性适合工业化加工的苛刻条件。

关键词: 黄曲霉毒素B₁, 枯草芽孢杆菌, 降解AFB₁酶组, 降解位点, 产物分析

Abstract:

This work aims to enrich the resources of biodegradable aflatoxin B₁（AFB₁）and to study the extracellular enzymatic properties of degrading AFB₁ by the screened Bacillus subtilis WTX1 as well as its degradation sites. Liquid chromatography tandem triple quadrupole mass spectrometry（UPLC-MS/MS）was used to detect the degradation solution by degrading three compounds with similar structures to the AFB₁ toxicity site. The two main extracellular enzymes degrading AFB₁ were Ea and Eb after chromatographic separation. The optimal degradation conditions: degradation enzyme Ea was in 50℃ and pH 5, and the degradation rate was up to 88.4%; degradation enzyme Eb was in 37℃ and pH 5, the degradation rate was 73.4%. The action sites of Ea enzyme were the double bond of furan ring, vanillin endo-ring and pentenone ring structure of AFB₁. The action sites of Eb enzyme were the vanillin endo-ring and pentenone ring structure of AFB₁. The degradation pathway of the enzyme group was via hydrolysis, hydrogenation and continuous removal of -CO group. In conclusion, the extracellular enzyme of strain WTX1 destroys the AFB₁ virulence sites by hydrolysis and thus plays a degradation role. The enzyme has good thermal stability, wide temperature and pH adaptability, which meet the harsh conditions of industrial processing.

Key words: aflatoxin B₁, Bacillus subtilis, AFB₁-degrading enzyme group, degradation sites, product analysis

杨冬, 唐璎. 枯草芽孢杆菌WTX1胞外酶降解AFB₁酶学特性及降解位点分析[J]. 生物技术通报, 2023, 39(4): 93-102.

YANG Dong, TANG Ying. Enzymatic Characterization and Degradation Sites of AFB₁ Degradation by the Extracellular Enzyme of Bacillus subtilis Strain WTX1[J]. Biotechnology Bulletin, 2023, 39(4): 93-102.

图/表 9

表1 质谱检测条件

Table 1 Mass detection condition

序号 Serial No.	目标物 Target	保留时间 Retention time/min	母离子 Parent ion/（m/z）	子离子 Daughter ion/（m/z）	去簇电压 Declustering voltage/DP	碰撞能量 Crash energy/CE
1	AFB₁	4.71	313.0	285.1*	140	31
1	AFB₁	4.71	313.0	240.9	140	49
2	香兰醛/ Vanillin	3.13	151.1	131.2* 81.1	120	32 41
3	5-羟甲基糠醛/5-（Hydroxymethyl）furfural	3.78	126.1	97.3* 41.2	120	35 49
4	1-戊烯-3-酮/1-Penten-3-one	2.85	56.1	27.2 57.1*	120	30 47

图1 不同变性处理对WTX1发酵上清液降解率影响不同小写字母表示不同处理间显著性差异（P<0.05）。下同

Fig. 1 Effects of different denaturation treatments on the degradation rates of WTX1 strain fermentation supernatant Different lowercase letters indicate significant differences between different treatments（P < 0.05）. The same below

图2 粗酶液Sephadex G-50凝胶层析

Fig. 2 Gel chromatography of crude enzyme liquid on Sephadex G-50

图3 AFB1降解酶离子交换层析峰值SDS-PAGE图

Fig. 3 SDS-PAGE graph of AFB1 degrading enzyme ion exchange chromatography peak value

图4 温度对降解率的影响

Fig. 4 Effect of temperature on the degradation

图5 发酵液pH值对降解率的影响

Fig. 5 Effect of fermentation liquid pH on the degradation

图6 金属离子对降解率的影响

Fig 6 Effects of metal ions on the degradation **：P<0.05；***：P<0.001

表2 酶对AFB1毒性位点相似结构化合物降解效果

Table 2 Degradation effects of enzymes on compounds with similar structures at the AFB1 toxicity site

酶 Enzyme	香兰醛 Vanillin/%	5-羟甲基糠醛 5-（Hydroxymethyl）furfural/%	1-戊烯-3-酮 1-Penten-3-one/%
Ea	92.4±1.6	42.8±1.8	86.3±4.2
Eb	67.8±3.6	83.2±3.2	0.6±2.4

图7 预测菌株WTX1降解AFB1机制

Fig. 7 Predictive mechanism of strain WTX1 degrading AFB1

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枯草芽孢杆菌WTX1胞外酶降解AFB₁酶学特性及降解位点分析

Enzymatic Characterization and Degradation Sites of AFB₁ Degradation by the Extracellular Enzyme of Bacillus subtilis Strain WTX1

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