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Kinetics of p-nitrophenyl Acetate Hydrolysis Catalyzed by Mucor javanicus Lipase in AOT Reverse Micellar Solutions Formulated in Different Organic Solvents

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Abstract

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W = ([water]/[AOT]) = 6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation.

Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.

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Abbreviations

AOT:

sodium 1,4-bis (2-ethylhexyl) sulfosuccinate

A 0 :

absorbance of the organic phase before extraction

A :

absorbance of the organic phase after extraction

CONICET:

Consejo Nacional de Investigaciones Cientificas y Técnicas

Conicyt:

Comisión Nacional de Investigaciones Científicas y Tecnológicas

Dicyt:

Dirección de Investigaciones Científicas y Tecnológicas

ε:

extinction coefficient

K p :

partition constant

(K M)app :

apparent Michaelis constant

K M :

Michaelis constant

Lip:

lipase

PNPA:

p-nitrophenyl acetate

PNP:

p-nitrophenol

TCE:

tetrachloroethylene

USACH:

Universidad de Santiago de Chile

V 0 :

initial reaction rate

V w :

volume of the organic phase

(V)corr :

corrected reaction rate

χ2 :

statistical parameter of the decay data fitting

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Acknowledgments

Thanks are given to Dicyt (USACH), CONICET and Conicyt (Grant # 1050058) for financial support.

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Authors and Affiliations

  1. Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40 - Correo 33, Santiago, Chile

    Elsa Abuin, Eduardo Lissi & Roxanna Duarte

  2. Departamento de Quimica, Universidad Nacional de Río Cuarto, Agencia Postal N°3 (5800), Rio Cuarto, Argentina

    M. Alicia Biasutti

Authors
  1. Elsa Abuin
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  2. Eduardo Lissi
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  3. M. Alicia Biasutti
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  4. Roxanna Duarte
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Correspondence to Elsa Abuin.

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Abuin, E., Lissi, E., Biasutti, M.A. et al. Kinetics of p-nitrophenyl Acetate Hydrolysis Catalyzed by Mucor javanicus Lipase in AOT Reverse Micellar Solutions Formulated in Different Organic Solvents. Protein J 26, 475–479 (2007). https://doi.org/10.1007/s10930-007-9087-y

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