133 Research Article Received: 7 May 2008 Revised: 19 June 2008 Accepted: 23 June 2008 Published online in Wiley Interscience: 26 August 2008 (www.interscience.com) DOI 10.1002/jctb.2018 Influence of carbohydrates on stability of papain in aqueous tetrahydrofuran mixture Andr ´ as Szab ´ o, a M´ arta Kotorm ´ an, a Ilona Laczk ´ o b and L M ´ aria Simon a* 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. c  2008 Society of Chemical Industry Keywords: stability of papain; carbohydrates; tetrahydrofuran; Schiff base formation INTRODUCTION The application of biocatalysts in non-conventional media has undergone significant development in the past two decades. 1–3 Proteases have been used with high efficiency for the synthesis of peptides and amino acid esters, furnishing stereo- and enantioselectivity in various aqueous organic solvents. Organic solvents exert considerable effects on the catalytic properties and stability of enzymes. The stability of enzymes is one of the most difficult problems in protein chemistry as a consequence of the large number of factors involved and the lack of experimental methods allowing an evaluation of their individual contributions. 4 The main disadvantage of using enzymes in organic media is that biocatalysts are less stable under these circumstances than in water. Several strategies may be applied for the stabilisation of enzymes: immobilisation, genetic engineering, chemical modification and the use of additives. 5–7 In a buffered aqueous environment, carbohydrates (mono- or disaccharides), glycerol and polyethylene glycol (PEG) are frequently used as polyhydroxy stabilisers, but the effects of these compounds at a molecular level are not known. 8 The heat stability and synthetic activity of papain are increased in the presence of glycerol, erythritol, xylitol and sorbitol, and a linear relationship is observed between the increase in the synthetic activity ratio and the overall concentration of hydroxy groups in the reaction media. 9 It is presumed that, probably by altering the hydration shell of an enzyme or by evolving protein–saccharide interactions, in water/organic solvent mixtures they can enhance the conformational stability of the enzyme. 10–12 Papain is a well-known sulfhydryl protease with various industrial applications. Its molecular structure comprises two distinct domains, with the active site in the groove between the domains. 13 As regards the distribution of ordered secondary structures, the first domain contains mainly α-helix, while the second domain has a large content of β -sheet and a lesser amount of α-helix. Water molecules present in ice-like networks play an important role in the stability of the enzyme, especially at the domain – domain interface. We earlier investigated the effects of water-miscible organic solvents (ethanol, 1,4-dioxane, acetonitrile (ACN) and tetrahydro- furan (THF)) on the hydrolytic activity and stability of papain. 14 THF proved to one of the most destabilising solvents. The aim of the present work was to study the effects of various saccharides (D-glucose, D-fructose, D-ribose, D-saccharose and D-raffinose) on the catalytic activity and stability of papain in a buffered water/THF (60%, i.e. 600 mL L −1 ) mixture (pH 8.0). The effects of these stabilis- ers on the structure of papain were followed by means of circular dichroism (CD) and fluorescence spectroscopic measurements. ∗ Correspondence to: L M´ aria Simon, Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics, University of Szeged, K¨ oz´ ep fasor 52, H-6726 Szeged, Hungary. E-mail: lmsimon@bio.u-szeged.hu a Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics, University of Szeged, K¨ oz´ ep fasor 52, H-6726 Szeged, Hungary b Institute of Biophysics, Biological Research Centre of Hungarian Academy of Sciences, Temesv´ ari krt. 62, H-6726 Szeged, Hungary J Chem Technol Biotechnol 2009; 84: 133–138 www.soci.org c  2008 Society of Chemical Industry