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Bounded water kinetic model of β-galactosidase in reverse micelles

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Abstract

In the present investigation, β-galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (Wo) were collected. It was observed that the usual kinetic model used for β-galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and Wo. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.

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Fig. 3a–e

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Abbreviations

A410:

Absorbance at 410 nm

AU:

Absorbance unit

AOT:

Concentration of Aerosol OT (M)

E :

Concentration of enzyme (mg/l)

E 0 :

Initial concentration of enzyme (mg/l)

ES :

Concentration of enzyme-substrate complex (mM)

ES′:

Concentration of galactosyl-enzyme complex (mM)

G :

Concentration of Galactose (M)

k 1 :

Rate constant for binding of enzyme and substrate (U/mg mM)

k −1 :

Rate constant for decomposition of enzyme-substrate complex (U/mg)

k cat :

Rate constant for elimination of p-nitrophenol from enzyme-substrate complex (U/mg)

k w :

Rate constant for binding of water and galactosyl-enzyme complex (U/mg M)

k −w :

Rate constant for binding of galactose and enzyme with concomitant elimination of water molecules (U/mg M)

K m :

Michaelis constant, as defined in Eq. 3 (mM)

N:

Number of experimental data points

ONPG:

o-nitrophenyl-β-D-galactopyranoside

P :

Concentration of o-nitrophenol (μM)

R i,exp :

Experimental data of specific initial activity of enzyme (U/mg)

R model :

Specific initial activity calculated from bounded water kinetic model (U/mg)

S :

Concentration of ONPG (mM)

U:

Reaction activity unit, defined as 1 μmol of molecules reacted or produced per minute

V app :

Apparent maximum specific activity as W o approaches infinity (U/mg)

W :

Concentration of free water (M)

W b :

Molar ratio of bounded water to AOT (−)

W o :

Molar ratio of total added water to AOT (−)

γ:

Coefficient of linear correlation (–)

ɛ:

Millimolar extinction coefficient (AU/mM cm)

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Acknowledgements

The author acknowledges partial financial support from TATUNG Co. and the National Science Council of the Republic of China under grant number NSC90-2214-E-036-008.

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Authors and Affiliations

  1. Department of Bioengineering, Tatung University, 40 Chungshan N. Rd., 3rd Sec., Taipei, Taiwan, Republic of China

    C. Will Chen & Chao-Chih Ou-Yang

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  1. C. Will Chen
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  2. Chao-Chih Ou-Yang
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Correspondence to C. Will Chen.

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Chen, C.W., Ou-Yang, CC. Bounded water kinetic model of β-galactosidase in reverse micelles. Bioprocess Biosyst Eng 26, 307–313 (2004). https://doi.org/10.1007/s00449-004-0367-z

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