A mathematical analysis of enzyme stabilization by a series‐type mechanism: Influence of chemical modifiers

Henley, James P.; Sadana, Ajit

doi:10.1002/bit.260260821

Cited by 56 publications

(15 citation statements)

References 31 publications

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“…An interpretation of these results can be made if we take into account the possible dynamic interactions of the more hydrophilic peptides with the hydrophilic amino acids of the [29,301 and hence could inhibit, to a certain extent, the transconformational mobility of the protein, leading to a higher activation energy of the reaction. Since the hydrophobic peptide arms do not affect the activation energy of the reaction, they seem unable to induce such direct interactions.…”

Section: Choice Of Peptidesmentioning

confidence: 97%

Effect of the microenvironment on the kinetic properties of immobilized enzymes

Bille¹,

Plainchamp²,

Lavielle³

et al. 1989

European Journal of Biochemistry

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A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co-polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages.The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions.The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.Immobilized enzyme technology underwent a wide development with the advent of numerous immobilization methods. This allowed fundamental studies of new aspects of enzyme-catalyzed reactions [I], but many applications in the industrial domain were also found [2, 31, mostly in bioconversion processes [4, 51 and in the analytical field [6, 71.Despite this large amount of work, the relationship between the immobilization method used and its effects on the properties of the immobilized enzyme is still difficult to predict [8], mainly because of changes due to the activation procedure and the effect on the microenvironment induced by the support [9]. One of the few approaches to this problem involves the study of the influence of a charged microenvironment on the activity of a-chymotrypsin [lo, 111. This work reports the effect of the charge of the microenvironment of the pK, of two groups present in the active center of the enzyme, through local modification of the pH.Because of this lack of information, we decided to perform a systematic study of the effect of the microenvironment on the properties of the enzyme. For this purpose, a new immobilization method had to be developed in which the influence of the chemical modification of the enzyme and of its microenvironment could be controlled. We developed a three-step strategy, in which the enzyme could be first activated, then inserted into a well-defined microenvironment and finally immobilized by multipoint covalent linkages. The model illustrated in this paper is for yeast alcohol dehydrogenase (ADH), Correspondence to J. Remade, Unite de Biochimie Cellulaire, FacultCs Universitaires de Namur, rue de Bruxelles 61, B-5000 Namur, Belgium Abbreviations. ADH, yeast alcohol dehydrogenase; SPDP, N-succinimidyl 3-(2-pyridyldithi...

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Section: Choice Of Peptidesmentioning

confidence: 97%

Effect of the microenvironment on the kinetic properties of immobilized enzymes

Bille¹,

Plainchamp²,

Lavielle³

et al. 1989

European Journal of Biochemistry

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“…The kinetics of enzyme inactivation were described assuming the existence of partially deactivated states (E 1 and E 2 ) with non-zero specific activity, where the k 1 and k 2 values are the first order deactivation coefficients in each case. The a i value is the percentage of specific activity of the enzymatic state E i with respect to the initial enzymatic state E (i01 or 2) (Henley et al 1984;Sadana et al 1987).…”

Section: Thermal Stabilitymentioning

confidence: 99%

“…The enzymatic deactivation curves with respect to temperature and co-solvent of both soluble and immobilized enzymes were analyzed using the twostep inactivation model reported by Henley and Sadana (1984), and were fitted to simple and double exponential decay models, with and without alpha, using the Enzfitter program.…”

Section: Thermal Stabilitymentioning

confidence: 99%

Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Giacomini

Irazoqui

Batista‐Viera

et al. 2007

Biocatalysis and Biotransformation

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Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzyme's thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three b-galactosidases from Escherichia coli , Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.

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“…The thermal deactivation curves of the soluble and immobilized enzyme derivatives were analyzed according to the two-step deactivation model proposed by Henley and Sadana (Henley and Sadana, 1984;Sadana and Henley, 1987):…”

Section: Thermal Stability At 50°cmentioning

confidence: 99%

Generating favorable nano‐environments for thermal and solvent stabilization of immobilized β‐galactosidase

Irazoqui

Villarino

Batista‐Viera

et al. 2002

Biotech & Bioengineering

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beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.

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A mathematical analysis of enzyme stabilization by a series‐type mechanism: Influence of chemical modifiers

Cited by 56 publications

References 31 publications

Effect of the microenvironment on the kinetic properties of immobilized enzymes

Effect of the microenvironment on the kinetic properties of immobilized enzymes

Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Generating favorable nano‐environments for thermal and solvent stabilization of immobilized β‐galactosidase

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