Abstract
BACKGROUND: The use of enzymes in organic solvents has extended the scale of their practical applications. Papain has been widely used in chemical syntheses because of its broad substrate specificity. The aim of the present study was to improve the stability of papain in aqueous tetrahydrofuran (THF) by using different saccharides. The effects of these carbohydrates on the structure of papain were followed by means of circular dichroism (CD) and fluorescence spectroscopic measurements.
RESULTS: In contrast with most organic solvents, 60% (i.e. 600 mL L^−1^) THF practically inactivated the enzyme within 30 min. Sugars protected papain from THF‐induced inactivation in the sequence D‐ribose > D‐fructose > D‐glucose > D‐saccharose > D‐raffinose. Ribose at 1.6 mol L^−1^ proved the most effective stabiliser: in 60% THF in the presence of ribose, papain preserved about 55% of its initial activity after 2 h. Fluorescence and near‐UV CD spectroscopic measurements revealed local changes in the papain conformation. With decrease in the free amino group content of the enzyme, protein‐carbohydrate interactions (Schiff base formation) were detected.
CONCLUSION: These results demonstrate that the catalytic activity and stability of papain may be increased in aqueous THF by using different carbohydrates, when a more compact structure of the enzyme is formed. Copyright © 2008 Society of Chemical Industry