Insecticide Detection through Protein Engineering of Nippostrongylus brasiliensis Acetylcholinesterase B

Abstract

The sensitivity of acetylcholinesterase (AChE) biosensors for insecticide detection could be increased substantially by engineering AChE B of Nippostrongylus brasiliensis. The introduction of 10 single and 4 double mutations into the AChE peptide chain led to an increase in sensitivity to 10 of the 11 insecticides tested. The combination of three mutants with the wild-type enzyme in a multienzyme biosensor array enabled the detection of 11 out of the 14 most important organophosphates and carbamates at concentrations below 10 microg/kg, the maximum residue limit of infant food. The detection limit for pirimiphos methyl could be reduced from 10 microg/L to a value as low as 1 ng/L (3.5 x 10(-)(12) mol/L). The newly created biosensors exhibited an extraordinary high storage stability. There was no loss of sensitivity of N. brasiliensis AChE B, immobilized on screen-printed, disposable electrodes, even after 17-month storage at room temperature.

Pestisit Analizlerinde Asetilkolinesteraz İnhibisyonuna Dayalı İletken Polimer Esaslı Biyosensörler

“…Pestisitler gibi inhibitörler varlığında aktif bölgede bulunan nükleofilik serin hidroksil grubu, pestisitte bulunan fosfor atomuna kovalent bağla bağlanır. Benzer durum karbamatların karbonil karbonu ile de yaşanır [11,12]. Dolayısıyla, organofosfat ve karbamatların tayini için asetilkolinesterazın inhibisyonu prensibi çok sık kullanılmaktadır [13][14][15][16].…”

High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification

“…The mutant NbAChE M301A was expressed in a Pichia pastoris X33 strain (Invitrogen, Karlsruhe, Germany) transformed with a pPICZaB vector (Invitrogen) bearing the Nippostrongylus brasiliensis AChE B gene (Hussein et al, 1999), with a single mutation causing an amino acid exchange at position 301 from methionine to alanine in the acyl pocket of the active site (Schulze et al, 2005). As a secretion signal the vector contained the Saccharomyces cerevisiae a-mating factor pre-pro leader sequence.…”

“…As a secretion signal the vector contained the Saccharomyces cerevisiae a-mating factor pre-pro leader sequence. Mutagenesis of the Nippostrongylus brasiliensis AChE B gene was performed by primer extension, as described elsewhere (Schulze et al, 2005).…”

“…Special AChEs of different origins were used as biological recognition elements in biosensors for the detection of neurotoxic insecticides (Schulze et al, 2003). To increase the sensitivity of AChE biosensors towards organophosphates and carbamates, we created a set of different single and double mutants of NbAChE B (Schulze et al, 2005). The replacement of methionine-301 in the acyl pocket of the active site by alanine (M301A) yielded a very sensitive mutant.…”

Ultrasensitive detection of organophosphate insecticides by carbon nanotube field-effect transistor

“…For rapid and low-cost detection of organophosphate and carbamate compounds, enzyme based methods has also been investigated. Several efforts involving the introduction of mutation in various AChEs by protein engineering have been made to increase the sensitivity of AChE biosensors toward many organophosphates and carbamates [4][5][6]. Schulze et al reported the highly sensitive detections of important insecticides at concentration below 10μg/kg, using biosensors composed of artificial AChE in combination with the amperometric transducers [6].…”