假蜜环菌黄曲霉毒素氧化酶的基因克隆、表达、纯化及酶学性质分析(英文)

Cloning,expression,purification and characterization of an aflatoxin-converting enzyme from Armillaria tabescens

【目的】黄曲霉毒素氧化酶(aflatoxin-oxidase,AFO)来源于假蜜环菌(Armillariella tabescens)的细胞内提取物,具有转化黄曲霉毒素B1(Aflatoxin B1,AFB1)的特性。为更进一步了解该酶的性质,我们克隆了AFO的基因,并进行了重组AFO蛋白的表达、纯化和酶学性质分析。【方法】本研究利用基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)获得的AFO短肽序列设计简并引物进行逆转录,再通过cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术获得了AFO基因的全长cDNA序列。构建重组表达载体pPIC9-afo,在毕赤酵母中进行重组AFO(rAFO)的融合分泌表达,用Ni离子螯合层析进行rAFO的纯化,获得有活性的rAFO后,对其进行肽质量指纹(peptide mass fingerprinting,PMF)鉴定和酶学性质分析。【结果】黄曲霉毒素氧化酶(AFO)基因的开放阅读框为2088 bp,编码695个氨基酸;肽质量指纹鉴定结果显示重组AFO的肽片段序列覆盖率为63.2%。活性测定表明纯化后的重组AFO(rAFO)比活力为234 U/mg;对rAFO进行酶学性质分析表明,对于底物黄曲霉毒素B1,rAFO的Km值为3.93±0.20×10-6 mol/L;反应最适温度为30℃,最适pH为6.0;30℃放置90 min后酶活力下降50%;rAFO在pH5.5-7.0之间酶活力较稳定,相对活力维持在51%-65%之间。【结论】本文第一次成功克隆并重组表达了一种具有黄曲霉毒素B1转化功能的酶——黄曲霉毒素氧化酶(aflatoxin-oxidase,AFO),纯化后的重组AFO(rAFO)具有较好的黄曲霉毒素B1转化活性,为进一步研究和应用奠定了基础。

[Objective] Aflatoxin B1（AFB1） is extremely mutagenic,toxic and a potent carcinogen both to humans and livestock.Aflatoxin-oxidase（AFO） was an aflatoxin-converting enzyme previously purified by us from Armillaria tabescens.In order to know better about the molecular characterization of this distinct enzyme,we expressed,purified and characterized the His6 tag fused aflatoxin-oxidase.[Methods] Based on sequences of peptides fragments of AFO previously obtained by Electrophoresis-Electrospray Ionization tandem mass spectrometry（ESI-MS/MS）,we cloned the cDNA of AFO using Switching Mechanism At 5′ end of the RNA Transcript（SMART） Rapid Amplification of cDNA Ends（RACE） technology and expressed this gene as a fusion protein in Pichia pastoris by using pPIC9-afo as vector.We purified the fusion enzyme using nickel affinity chromatography.We identified the recombinant aflatoxin-oxidase（rAFO） by both western blot and peptide mass fingerprinting（PMF）.Moreover,we characterized several enzymatic properties of the rAFO using AFB1 as the substrate including Km value,optimum temperature,optimum pH,thermal stability and pH stability.[Results] The AFO gene is 2321 bp long with a coding region of 2088 bp encoding 695 amino acids.Peptide mass fingerprinting（PMF） identification showed a 63.2% coverage of the molecule compared to the theoretical tryptic cleavage of the rAFO.The recombinant aflatoxin oxidase was purified 5.99-folds using nickel affinity chromatography.It has a specific activity of 234 U/mg.Kinetics studies showed that the rAFO converted AFB1 with the Km value of 3.93±0.20×10-6 mol/L under its optimal conditions of pH6.0 and 30℃.Thermostability investigation revealed that the rAFO had a half-life of 90 min at 30℃,and pH stability results suggested that the rAFO was relatively stable when pH ranged from 5.5 to 7.5.[Conclusion] It appears to be the first successful production of the recombinant aflatoxin oxidase（rAFO） with AFB1-converting ability from Armillaria tabescens.The purified rAFO with preferably AFB1-converting activity confirms that this recombinant aflatoxin oxidase is now ready for further studying.