How do additives affect enzyme activity and stability in nonaqueous media?

✍ Scribed by Angeliki Öste Triantafyllou; Ernst Wehtje; Patrick Adlercreutz; Bo Mattiasson

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 159 KB
• Volume: 54
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19970405)54:1<67::aid-bit8>3.0.co;2-w

✦ Synopsis

The catalytic activities of lyophilized powders of ␣-chymotrypsin and Candida antarctica lipase were found to increase 4-to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation. Increasing amounts of sorbitol in the chymotrypsin preparation produced a modest increase in activity. The additives are basically thought to serve as immobilization matrices, the sorbitol being inferior because of its poor mechanical properties.

Besides their role as supports, the buffer species were indispensable for the transesterification activity of chymotrypsin because they prevented perturbations of the pH during the course of the reaction. Hence, increasing amounts of buffer species yielded a 100-fold increase in transesterification activity. Effects of pH changes were not as predominant in the peptide synthesis and the lipase-catalyzed reactions.

Immobilization of the protease on celite resulted in a remarkable improvement of transesterification activity as compared to the suspended protease, even in the absence of buffer species. Immobilization of the lipase caused a small improvement of activity. The activity of the immobilized enzymes was further enhanced 3-4 times by including increasing amounts of buffer salts in the preparation.

The inclusion of increasing amounts of sodium phosphate or sorbitol to chymotrypsin rendered the catalyst more labile against thermal inactivation. The denaturation temperature decreased with 7°C at the highest content of sodium phosphate, as compared to the temperature obtained for the denaturation of the pure protein.

The apparent enthalpy of denaturation increased with increasing contents of the additives. The enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results.