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Abstract

Background: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8 th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein.

Insecticide Detection through Protein Engineering of Nippostrongylus brasiliensis Acetylcholinesterase B

“…Several approaches were performed to increase the stability of AChE, for example, by adding protective compounds or, in the case of the D melanogaster AChE, by protein engineering. 54 The exchange of a cysteine residue, which is not involved in an intra-or intermolecular disulfide bridge, by valine increased the temperature stability of the D. melanogaster AChE.…”

“…The negative effect of moisture on the stability of AChE was already reported elsewhere. 54,55 We also tested the reactivation of the biosensor, a further criterion for its functionality. The AChE activity of the new and 17-month-old biosensors could be completely reactivated after each measurement using 2-PAM.…”

Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells

“…Similarly the free Cys residues in herpes simplex virus type 1 glycoprotein D [24] and in human granulocyte colony stimulating factor [25] , are not necessary for their activities and are located in inaccessible positions, in the transmembrane region and in the interior of the protein, respectively. It has been reported that elimination of a free Cys residue in human interferon-β and in Drosophila acetylcholinesterase improves their stability [26] , [27] . Furthermore, it is known that disulfide isomerization driven by a free Cys residue is required to activate phage endolysin [28] , [29] .…”