THE PRESSURE-DEPENDENCE OF 2 BETA-GLUCOSIDASES WITH RESPECT TO THEIR THERMOSTABILITY

A comparative study of temperature and pressure effects were carried o ut by using two homologous enzymes exhibiting different thermostabilit y and oligomery: almond beta-glucosidase and Sulfolobus solfataricus b eta-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 b eta-glucosidase, the moat pressure sensitive enzyme of the two was con tinuously affected by pressure up to 1.5 kbar. Activation volume chang es revealed that the inactivation of almond beta-glucosidase was due t o both catalytic step inactivation and enzyme-substrate binding inacti vation. Structural modifications generated by pressure, related to a l oss of activity did not affect the global conformation of almond beta- glucosidase, after depressurization. In contrast, Sulfolobus solfatari cus beta-glucosidase was a highly barostable enzyme. It maintained a h alf-life of 91 h at 60 degrees C and 2.5 kbar. Moreover, this enzyme a ppeared to be activated by pressure between atmospheric pressure and 2 .5 kbar with a maximal activity at 1.25 kbar, However, this enzyme sti ll displayed the best catalytic efficiency at atmospheric pressure bec ause of a K-m value drastically increased by pressure. Activation volu me changes indicated that the hydrolysis reaction catalysed by Sulfolo bus solfataricus beta-glucosidase, was alternatively favoured and disf avoured by pressure due to the catalytic step activation or inactivati on associated with the enzyme-substrate binding step being continuousl y inactivated by pressure.