재사용가능한 불균일상 바이오촉매

Abstract

We propose a stable method for immobilizing enzyme on the support with a unique strategy by tagging a sequence intothe enzyme that we want to attach onto a beadsupport. Importantly, we can do this for any enzyme. Here, we chose N-methyl tryptophan oxidase and alkaline phosphatase for analyzing the reliability of site-specific immobilization on the coated magnetic beads. In this study, we showed that site-specifically immobilized enzymes have advantages compared to free enzymes. We could achieve site-specific modification of protein onto the surface of special amine coated magnetic beads, which we analyzed in detail. Immobilized enzyme was stable after a long time of storage, even after 1 year. In addition, we found that it can be used as a portable, colorimetric detection system for L-abrine, a biomarker of toxic protein abrin poisoning. The detection limit of our sensor was as low as 4μM when using UV/Vis spectrophotometer and similar limits were detectable by eye. We show that our approach is feasible in real world samples by proving the detection capabilities with mock urine. Finally, our sensor system was also found to be portable owing to its heat stability thereby eliminating the need for refrigeration duringcarrying or storage.|본 연구에서는 효소에 아미노산 시퀀스를 도입함으로써 효소를 supporting material에 안정하게 고정시키는 독특한 방법을 제시한다. 중요한 것은 이 방법은 어느 효소에도 적용이 가능하다는 점이다. 여기서 코팅된 자성입자 위에 위치특이적으로 효소를 고정화시키는 것이 얼마나 믿을만한 방법인지 분석하기 위해서 우리는 N-메틸트립토판 산화효소(N-methyl tryptophan oxidase)와 알칼리성 인산분해효소(alkaline phosphatase)라는 효소를 선택하였다. 이 연구에서 우리는 위치특이적(site-specifically)으로 고정된 효소가 보통의 효소보다 장점이 많다는 것을 보여주었다. 우리는 아민(amine)으로 코팅된 자성입자의 표면을 단백질로 위치특이적인 수정이 가능하다는 것을 자세하게 분석하였다. 고정화된 효소는 1년이라는 장시간의 보관후에도 안정된 성능을 보였다. 또한, 우리는 우리의 고정화된 효소가 위험한 독성단백질인 아브린(abrin)의 바이오마커인L-아브린(L-abrine)의 휴대가능한 발색이 있는 검출 시스템(colorimetricdetection system)으로 아브린에 중독, 노출되었을 시 사용가능한 것을 알아내었다. 본 센서의 검출한계는 UV/Vis spectrophotomter를 사용했을 때 4μM까지였고, 사람의 눈으로도 비슷한 한계까지 볼 수 있었다. 실제에서도 우리의 연구가 적용이 가능하다는 것을 실험용 소변에서의 검출능을 증명했다. 끝으로, 우리의 센서는 열적 안정성 덕분에 이동하는 동안의 별도의 냉장기구의 필요성을 제거했기 때문에 휴대가 가능한 센서라는 것을 알 수 있었다.; We propose a stable method for immobilizing enzyme on the support with a unique strategy by tagging a sequence intothe enzyme that we want to attach onto a beadsupport. Importantly, we can do this for any enzyme. Here, we chose N-methyl tryptophan oxidase and alkaline phosphatase for analyzing the reliability of site-specific immobilization on the coated magnetic beads. In this study, we showed that site-specifically immobilized enzymes have advantages compared to free enzymes. We could achieve site-specific modification of protein onto the surface of special amine coated magnetic beads, which we analyzed in detail. Immobilized enzyme was stable after a long time of storage, even after 1 year. In addition, we found that it can be used as a portable, colorimetric detection system for L-abrine, a biomarker of toxic protein abrin poisoning. The detection limit of our sensor was as low as 4μM when using UV/Vis spectrophotometer and similar limits were detectable by eye. We show that our approach is feasible in real world samples by proving the detection capabilities with mock urine. Finally, our sensor system was also found to be portable owing to its heat stability thereby eliminating the need for refrigeration duringcarrying or storage.