假蜜环菌醛酮还原酶AtAKR297克隆表达及对AFB1转化作用

Armillariella tabescens aldo-keto reductase AtAKR297: cloning, expression, and application in reduction of aflatoxin B1

摘要:

【背景】黄曲霉毒素B₁ (aflatoxin B₁, AFB₁)是迄今为止发现的毒性最强的生物毒素之一，它通过污染花生、玉米、棉花和辣椒等不同作物，对人类和动物健康造成严重威胁。由于醛酮还原酶能够将多种醛酮类物质还原为毒性更小的醇类物质从而发挥解毒作用，所以推测醛酮还原酶可能具有转化AFB₁的可能。【目的】克隆了假蜜环菌(Armillariella tabescens)中的醛酮还原酶AtAKR297，以研究醛酮还原酶AtAKR297对AFB₁的转化作用。【方法】使用TRIzol法提取假蜜环菌的总RNA，根据蛋白质谱获取的肽段信息及cDNA末端快速克隆(rapid-amplification of cDNA ends, RACE)技术获取醛酮还原酶AtAKR297基因全长序列；构建醛酮还原酶AtAKR297的表达系统，用亲和层析对蛋白进行纯化，并进行酶活力检测及酶学性质分析。在pH 8.0缓冲体系中，将醛酮还原酶AtAKR297与AFB₁反应12 h，用高效液相色谱仪对残余的AFB₁进行检测，并用高分辨率液相色谱-质谱仪对转化产物进行鉴定。【结果】成功克隆出假蜜环菌的全长AtAKR297基因，基因长度为894 bp，共编码297个氨基酸；pET28a(+)-AtAKR297质粒转入大肠杆菌(Escherichia coli) BL21(DE3)宿主菌中，实现诱导表达并对酶学性质进行分析。AtAKR297的最适反应温度为20 ℃，最适反应pH值为8.0；在15-50 ℃具有较好的稳定性，孵育30 min酶活可维持60%以上；在pH 5.0-8.0具有较好的稳定性，孵育2 h酶活可维持75%以上。醛酮还原酶AtAKR297与AFB₁的相互作用质谱鉴定结果显示AtAKR297能作用于AFB₁ (依赖NADPH)，将AFB₁还原为毒性降低的黄曲霉毒醇(aflatoxicol, AFL)，该产物分子式为C₁₇H₁₄O₆。【结论】成功从假蜜环菌中克隆出醛酮还原酶基因AtAKR297，并构建了具有醛酮还原酶活性的AtAKR297的原核表达系统。AtAKR297在NADPH的存在下能将AFB₁还原为毒性降低的AFL。

Abstract:

[Background] Aflatoxin B₁ (AFB₁) is one of the most potent biotoxins discovered to date, posing threats to the health of humans and animals by contaminating various crops such as peanut, maize, cotton, and chili pepper. Given that aldo-keto reductases can detoxify a range of aldehydes and ketones by reducing them to less toxic alcohols, it is hypothesized that these enzymes may have the potential to convert AFB₁. [Objective] To investigate the role of the aldo-keto reductase AtAKR297 cloned from Armillariella tabescens in the conversion of AFB₁. [Methods] The TRIzol method was employed to extract the total RNA from A. tabescens, and the full-length sequence of the AtAKR297 gene was obtained based on the peptide information from proteomic analysis and RACE. An expression system for AtAKR297 was constructed, and the expressed protein was purified by affinity chromatography. The enzyme activity and kinetic properties were then analyzed. AtAKR297 was reacted with AFB₁ in a buffer system at pH 8.0 for 12 h. The residual AFB₁ was determined by HPLC, and the conversion products were identified by high-resolution LC-MS. [Results] The full-length AtAKR297 gene from A. tabescens was successfully cloned, with a length of 894 bp, encoding 297 amino acid residues. The recombinant plasmid pET28a(+)-AtAKR297 was transformed into Escherichia coli BL21(DE3) cells for induced expression of the target protein. The enzymatic properties of the expressed protein were then characterized. AtAKR297 showed the highest activity at 20 ℃ and pH 8.0. It showcased good thermal stability at 15-50 ℃, with the relative activity maintained above 60% after incubation for 30 min. In addition, AtAKR297 demonstrated good stability at pH 5.0-8.0, with the relative activity maintained above 75% after incubation for 2 h. The MS results of the interaction between AtAKR297 and AFB₁ showed that AtAKR297 reduced AFB₁(dependent on NADPH) to less toxic aflatoxicol (AFL, C₁₇H₁₄O₆). [Conclusion] The aldo-keto reductase gene AtAKR297 was successfully cloned from A. tabescens, and a prokaryotic expression system with aldo-keto reductase activity was constructed. AtAKR297 can reduce AFB₁ to less toxic AFL in the presence of NADPH.