Effects of Modulation on Thermal Inactivation

Thermal inactivation of enzymes is usually evaluated under non-reactive conditions. However, it is a well-reported fact that enzyme stability is different under reactive conditions (Villaume et al. 1990 Ospina et al. 1992), suggesting that substrates and products of reaction affect it. Results obtained under non-reactive conditions may then poorly predict the actual stability of the enzyme within the bioreactor. It has been frequently reported that the substrate protects the enzyme from thermal inactivation. Substrate protection was first modeled by O Neill (1972) and then redefined in terms of the so-called protection factor by Chen and Wu (1987). We have generalized this concept in terms of modulation factors proposing that any substance that interacts with the enzyme during biocatalysis, is a potential modulator (M) of its stability and that such modulation can be positive (protection) or negative (destabilization) (Illanes et al. 1994). According to that hypothesis, all enzyme species (free enzyme and enzyme complexes) will inactivate at different rates so that  

The modulation factor of substance M in the inactivation stage i is then defined as  

Positive modulation (protection) by substrate has been explained in terms of the stiffness of the enzyme structure that it promotes (Villaume et al. 1990). Negative modulation (destabilization) has also been reported for substrate (Illanes et al. 1998a) and other catalytic modulators (Alvaro et al. 1991). This behavior can be explained in terms of the promotion of quaternary structure dissociation (Misset 1993) and alteration of the oxidation stage in the active site of the enzyme (Bourdillon et al. 1985). 

As an example. Fig. 5.15 shows the kinetics of reaction and inactivation of chitin-immobilized (3-galactosidase at 27.5°C (Illanes et al. 2000). In this case the enzyme is competitively inhibited by the product galactose but not for glucose and inactivation for all enzyme species was modeled according to a two-stage series mechanism 

A similar study was conducted with immobilized penicillin acylase in this case, both the substrate (penicillin G) and the non-competitive inhibitor product (6-aminopenicillanic acid) were positive modulators, while the competitive inhibitor product (phenylacetic acid) was a negative modulator of enzyme stability (Illanes et al. 1996) again all compounds that interacted with the enzyme during catalysis were modulators of enzyme stability. 

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