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

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

摘要:

摘要: 【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。

Abstract:

Abstract: ［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．