Are Associated Ions Important for Biocatalysis in Organic Media?

Abstract

The nature of the buffer species used in the drying process is important when lyophilized enzyme preparations are suspended in organic media. The activity of subtilisin Carlsberg in a transesterification reaction was found to vary depending on the nature of the buffer used. It was postulated that the large excess of salt present in the dried powder could be affecting enzymatic activity by alterations to the microscopic structure of the powder. To establish if this were true, microscopic changes were eliminated by covalently immobilising the enzyme onto a macroporous polymer support so that the counter-ions could be exchanged by washing with dilute salt solutions. It was found that in the immobilised samples no significant effects of salt ions were noted. This was the case even when salt ions were in considerable excess of that needed to balance protein charges. Hence the activity variations noted in freeze-dried powders are probably due to changes to the microscopic structure, rather than to molecular scale interactions. Similarly the previously observed activating effect of crown ether solutions on freeze-dried powders is not repeated on an immobilised preparation suggesting that this too may be due to a microscopic effect on the powder.

Key Words:

• subtilisin Carlsberg
• organic solvent
• immobilisation
• counter-ions