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Determination of esterase activity

GP Gabriella Cavazzini Pavarina
EL Eliana Gertrudes de Macedo Lemos
NL Natália Sarmanho Monteiro Lima
JJ João Martins Pizauro Jr.
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The esterase activity was determined discontinuously in a spectrophotometer at 405 nm. The reaction was initiated by adding 20 µL of the enzyme at 0.1 mg/mL to 0.1 M sodium acetate buffer at pH 6.5 and 1 mM p-nitrophenyl acetate for 15 min at 37 °C. Substrate affinity was determined using the following substrates (1 mM, Sigma-Aldrich): p-nitrofenyl acetate, p-nitrofenyl butyrate, p-nitrofenyl valerate, p-nitrofenyl octanoate, p-nitrofenyl dodecanoate, p-nitrofenyl myristate, and p-nitrofenyl palmitate. One unit of enzymatic activity was defined as 1 µmol p-nitrophenol released per minute per mg of protein under previously established standard assay conditions. The tests were performed in triplicate, and each reaction included a control without the enzyme to measure the spontaneous hydrolysis of the substrate.

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