The pressure-dependence of two beta-glucosidases with respect to their thermostability.
A comparative study of temperature and pressure effects were carried out by using two homologous enzymes exhibiting different thermostability and oligomery: almond beta-glucosidase and Sulfolobus solfataricus beta-glucosidase. Both the activity and stability were studied using an in-house built bioreactor allowing injection, stirring, sampling and on-line spectrophometric monitoring with retention of pressure up to 2.5 kbar and temperature control possible up to 150 degrees C. Almond beta-glucosidase, the most pressure sensitive enzyme of the two was continuously affected by pressure up to 1.5 kbar. Activation volume changes revealed that the inactivation of almond beta-glucosidase was due to both catalytic step inactivation and enzyme-substrate binding inactivation. Structural modifications generated by pressure, related to a loss of activity did not affect the global conformation of almond beta-glucosidase, after depressurization. In contrast, Sulfolobus solfataricus beta-glucosidase was a highly barostable enzyme. It maintained a half-life of 91 h at 60 degrees C and 2.5 kbar. Moreover, this enzyme appeared to be activated by pressure between atmospheric pressure and 2.5 kbar with a maximal activity at 1.25 kbar. However, this enzyme still displayed the best catalytic efficiency at atmospheric pressure because of a Km value drastically increased by pressure. Activation volume changes indicated that the hydrolysis reaction catalysed by Sulfolobus solfataricus beta-glucosidase, was alternatively favoured and disfavoured by pressure due to the catalytic step activation or inactivation associated with the enzyme-substrate binding step being continuously inactivated by pressure.
Hamon
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Laboratoire de Technologie Enzymatique, URA 1442 CNRS, Université de Technologie de Compiègne, France.
Dallet
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Legoy
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D003300:Copper; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D009754:Nuts / Q000201:enzymology; D011312:Pressure; D016944:Sulfolobus / Q000201:enzymology; D013816:Thermodynamics; D013997:Time Factors; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism*
D046911:Macromolecular Substances; D003300:Copper; D001617:beta-Glucosidase