Construction Of Immobilized-AChE Column And Its Application In Screening Active Constituents From Natural Products

The target enzymes play an important roles in the research and development of new pesticides, and most of the targets of highly active insecticides are key enzymes in the metabolism of insect. Immobilization of target enzymes has broad prospects in rapid screening of active constituents from complex system for its easier purification, higher recovery, and simple, reproducible operation process. In pesticide research field, it is mainly used as biosensors to detect pesticide residues in environment or to degrade corresponding pesticide.However, there are few studies which applied the immobilization of target enzyme for screening of the inhibitors. As we all konw, acetylcholinesterase is a key enzyme that involved in neural transmission of animals and insects, and therefore it is the target of various neurotoxicants. In this paper, an economical and effective immobilized-AChE column was prepared using sol-gel embedding method for screening the AChE inhibitor and insecticidal compounds from complex natural products.In this study, two methods of immobilizing AChE, sol-gel crosslinking and sol-gel embedding, were first optimized to construct immobilized-enzyme columns. The reaction conditions including the effect of different silicate ester, dosage, temperature and gel’s swelling rate had been investigated systematically. Electron scanning microscopic (ESM) analysis and FT-IR analysis was used to characterize complex carrier. Then, the immobilized-enzyme column was combined with HPLC to establish a model system that could rapidly detect and isolate insecticidal constituents from complex natural products. AChE from electric eel was immobilized using optimized sol-gel methods and the enzyme activity recovery rates were 71.3±8.1%(Km=27.24±0.13 mmol L-1) for sol-gel crosslinking and 82.8±6.0%(Km=26.16±0.09 mmol L-1) for sol-gel embedding.By comparing immobilized enzyme chromatogram column with black carrier column, it was found that the components which were absorbed by immobilized enzyme were this enzyme’s inhibitors. For example, magnolol (1) and honokiol (2), were isolated from the ethanol extract of Magnolia officinalis with the IC50 values of 18.32±2.01 mg L-1 and 15.19±3.89 mg L-1, respectively. The data also indicate that its mode of AChE inhibition belonged to the mixed type. While those components which were not absorbed by immobilized enzyme usually had not inhibitive activity, such as, quercetin-3-O-I-rhamnoside and (3) dihydrokaempferol-3-O-Z-rhamnopyranoside (4). In vivo bioassay indicated 1 and 2 showed insecticidal activity against Nilaparvata lugens with the LC50 values of 86.32 and 36.43 mg L"1 comparable to that of commonly used insecticide chlorpyrifos (81.72 mg L-1). Moreover, molecular docking was carried out against the homology model of N. lugens AChE. The complexes showed that 1 and 2 placed themselves nicely into the active site of the enzyme and exhibited interaction energy which was in accordance with our activity profile data.Using this screening method, we also got flavipin, which came from secondary metabolite of endophytic fungi CDW7 in Ginkgo biloba. The IC50 value of flavipin (5) was 30.93±1.21mg L-1。...