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