Use of EST2 from Use of EST2 from A. acidocaldariusA. acidocaldarius as bioactive part in as bioactive part in biosensors for organophosphates detectionbiosensors for organophosphates detection

Ferdinando Febbraio

Institute of Protein Biochemistry of CNR, Italy

OP high doses are acutely neurotoxic

OP low doses have several suspected mechanisms of

action, including disruption of nuclear transcription factors,

interference with neural cell development and

neurotransmitter systems, and altered synaptic formation.

GC-MSHPLC-MSUPLC-ESMS

ExpensiveInsufficient for in situ and real-time detectionUnable to provide any information concerning the toxicity of the sampleRequire highly trained personnel

High resolutionAutomationIdentification of a large number of metabolite in a single measure

AChE biosensors

Comparable or even better analytical performances than traditional systemsSmall, cheap and fast.Easy to handle (do not require trained technicians to be used)Real-time detection

Not sufficiently robust to deal with raw samplesDo not offer adequate selectivity

Esterase 2 from Esterase 2 from Alicyclobacillus acidocaldariusAlicyclobacillus acidocaldarius

Manco et al., Biochem. J. 1998 Manco et al., Biochem. J. 1998

EST2 is a monomeric carboxylesterase of about 34 EST2 is a monomeric carboxylesterase of about 34 Kda belonging to the hormone sensitive lipase (HSL) Kda belonging to the hormone sensitive lipase (HSL) family.family.EST2 hydrolyses monoacyl esters of diEST2 hydrolyses monoacyl esters of difffferent acyl erent acyl chain lengths, displaying maximal activity on chain lengths, displaying maximal activity on pp--nitrophenyl esters characterized by an acyl chain nitrophenyl esters characterized by an acyl chain length of 6length of 6--8 carbon atoms, at an optimal 8 carbon atoms, at an optimal temperature of 70 °C.temperature of 70 °C.

ki

(k2)K

i

k cat

(k3)

k transKD

H2O

E + I E•I E-I

E + I-OH

E-I• Idrossilammina

Idrossilammina

E + I- Idrossilammina

Febbraio et al., Extremophiles, 2008Febbraio et al., Extremophiles, 2008

IKKS

KkA

im

iic

/1

Febbraio et al., Analitical Chemistry, 2011Febbraio et al., Analitical Chemistry, 2011

4-Methylumbelliferyl butyrate4-MethylumbelliferoneExcitation wavelength: 365 nm

Emission wavelength: 445 nm

Binding with fluorescent probes

Characterization of newEST2 mutants

Development of principalcomponent analysis software

for OP mixture detection

Assay on nerve agents

PeoplePeople

Paola CarulloPaola CarulloGiovanni P. CetrangoloGiovanni P. CetrangoloCarla GoriCarla GoriGiuseppe MancoGiuseppe MancoLuigi MandrichLuigi Mandrich

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