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Enzyme Research
Volume 2019, Article ID 6139863, 9 pages
https://doi.org/10.1155/2019/6139863
Research Article

Acetylcholinesterases from Leaf-Cutting ant Atta sexdens: Purification, Characterization, and Capillary Reactors for On-Flow Assays

1Federal University of São Carlos, Department of Chemistry, São Carlos, SP, Brazil
2São Paulo University, Instituto de Ciências Biomédicas (ICB), São Paulo, Brazil
3São Paulo State University, Center for the Study of Social Insects, Rio Claro, SP, Brazil

Correspondence should be addressed to Quezia B. Cass; rb.racsfu@ssacq and Dulce Helena F. Souza; rb.racsfu@eclud

Received 8 February 2019; Accepted 12 May 2019; Published 1 July 2019

Academic Editor: Qi-Zhuang Ye

Copyright © 2019 Adriana M. Dos Santos et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Acetylcholinesterase (AChE) is responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) leading to acetate and choline (Ch) release. The inhibition of AChE produces a generalized synaptic collapse that can lead to insect death. Herein we report for the first time the isolation of two AChEs from Atta sexdens which were purified by sulphate ammonium precipitation followed by ion exchange chromatography. AsAChE-A and AsAChE-B enzymes have optimum pH of 9.5 and 9.0 and higher activities in 30/50°C and 20°C, respectively, using acetylthiocholine (ATCh) as substrate. Immobilized capillary enzyme reactors (ICERs) were obtained for both enzymes (AsAChE-A-ICER and AsAChE-B-ICER) and their activities were measured by LC-MS/MS through hydrolysis product quantification of the natural substrate ACh. The comparison of activities by LC-MS/MS of both AChEs using ACh as substrate showed that AsAChE-B (free or immobilized) had the highest affinity. The inverse result was observed when the colorimetric assay (Elman method) was used for ATCh as substrate. Moreover, by mass spectrometry and phylogenetic studies, AsAChE-A and AsAChE-B were classified as belonging to AChE-2 and AChE-1 classes, respectively.