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

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.

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.

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 x 10(-6) mol/L under its optimal conditions of pH 6.0 and 30 degrees C. Thermostability investigation revealed that the rAFO had a half-life of 90 min at 30 degrees C, and pH stability results suggested that the rAFO was relatively stable when pH ranged from 5.5 to 7.5.

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.