Improvement Of The Expression Of Recombinant Drosophila Melanogaster Acetylcholinesterase In P. Pastoris And Of The Derived New Biosensor For Fast Detection Of Pesticide Residues

The acetylcholinesterase (AChE)-based methods are ubiquitously used for rapid detection and regulation of organophosphate and carbamate pesticide residues in Chinese markets. However, there are still two major drawbacks hampering the application of this method: firstly,the sensitivity of the enzyme toward pesticides is not desirable in the term of the related legislation; secondly, most of these tests are based on colorimetric method which is often interfered by pigments in real samples.To overcome these, we optimized and synthesized the cDNA of DmAChE based on the preferred codon usage of P.pastoris by over-lapping PCR. The modified DmAChE cDNA fragment was then ligated to P.pastoris expression vector—pPIC9K under the control of the alcohol oxidase gene AOX1 promoter. Via electroporation and MM plate fast screening, one transformant expressing AChE with high activity was obtained. In shake-flask after induction by methanol, this strain secreted AChE activity at high level of 718 U/mL, 3.4times higher than the strain we previously obtained with original DmAChE cDNA.Based on the high sensitive R-DmAChE harvested, we applied it into electrochemical biosensor, introducing MWNTs to improve its performance in several aspects. We employed Prussian blue as electron mediator by electrochemical polymerization on MWNTs to reduce the oxidation potential of thiocholine, the enzymatic product, to 200 mV in order to eliminate interference caused by high oxidation potential. The enzyme was immobilized by glutaraldehyde in addition to MWNTs. With high surface-to-volume ratio, MWNT not only significantly increased the amount of Prussian blue polymerization on the electrode, but also increased the signal currents by 40% and reduced the response time of the sensor by over 300%. Besides the advantage mentioned above, MWNTs improved the stability of the sensor and increased the sensitivity to carbofuran by 10 fold, duo to its unique amphiphilicmicro-environment. The optimized sensor can detect 0.5μg/L of dichlorvos and 5μg/L of carbofuran in four spiked beverages, which illustrated that the sensor could resist the interference of caramel, alcohol, vitamin and protein.In conclusion, the recombinant enzyme obtained in this study could be a high-quality and affordable raw material for fast detection of pesticide residual in China; and the Anti-interference AChE-based biosensor boosted by MWNT could be a promising tool for fast pesticide residual detection.