Keywords: Araujia hortorum Fourn., organic solvents, plant protease, substrate preferences, thermostability.

Most enzyme catalytic processes are carried out in aqueous solutions. Nevertheless, numerous investigations have demonstrated that the enzymes can be active in other media such as organic solvents, gaseous solvents and supercritical fluids.

In many cases, water constitutes a little appropriate solvent for the chemical reactions, since many substrates of commercial interest has low solubility in water and they are frequently unstable in aqueous solution. Besides, several products of the enzyme catalytic processes can not be obtained in aqueous media due to their unfavourable equilibrium of reaction. For these reasons, there is an increasing interest to carry out catalyst processes in other media, such as organic solvents. 

The suspension or dissolution of enzymes into non-aqueous media offers other advantages over the use of enzymes in water, such as the reduction of the inhibition due to substrates and/or products, the easy recovery of product and biocatalyst, the increase of the thermostability and the change of the substrate specificity of some enzymes. These facts have extended the use of enzymes as highly specific catalysts for the resolution of racemic mixtures, oxidation of steroids and synthesis of esters and peptides, among other applications.

Unfortunately, there are also some drawbacks, such as the formation of secondary products, hydrolysis of the substrates and products and changes in the enzyme activity and stability. However, the use of different strategies such as the enzyme immobilization and media and enzyme engineering are reducing these limitations.

One of the most important parameters to be considered in the use of organic media is the water content because it can change the enzymes properties in such media. Thus, in order to study the effect of organic solvents on the behaviour of an enzyme, it is necessary to fix constant water content on the enzyme.

In this paper we studied the effect of different organic solvents (1-octanol, trichloroethylene, ethanol, ethyl acetate, tetrahydrofuran, cyclohexane, propanone, acetonitrile, dichloromethane, chlorobenzene, N,N-dimethylformamide (DMF), acetophenone, diethyl ether, methanol, ethylene glycol and toluene) with low and constant water content on substrate preferences, thermostability and stability (caseinolytic activity retention after 4 hrs) of proteases of Araujia hortorum Fourn. (Asclepiadaceae).

Araujiain is the crude enzyme preparation obtained from the latex of fruits of Araujia hortorum Fourn. (Asclepiadaceae). This preparation contains cysteine proteases belonging to the papain family.

In order to carry out the study of the stability of araujiain in organic media with low water content, a statistical design by clustering 70 organic solvents according to their physico-chemical properties extracted from the literature was carried out, and one representative organic solvent of each group was chosen for the study. 

The determination of the water amount in both organic solvent and enzyme after incubation in organic media with different water concentration was carried out by the optimized Karl Fisher method. The enzyme water amount was expressed as a relative fraction of water (relation between the molar concentration of water in the enzyme and the molar concentration of the pure water), and this value was fixed in 0.5.

The araujiain stability in organic media with low water content was measured as residual activity after 4 hrs of incubation at 40ºC, using casein as substrate. An arbitrary enzyme unit (Ucas) was defined as the amount of protease which produces an increment of one absorbance unit per min in the assay conditions. Moreover, the thermal behaviour of araujiain was evaluated by measuring the residual caseinolytic activity after incubation of the sample in Tris-HCl buffer (pH 8), in ethanol and DMF at 40, 50 and 65ºC at periodical intervals of time.

The substrate preferences were determined as esterolytic activity of araujiain using N-α-carbobenzoxy-p-nitrophenyl esters of some amino acids (Gln, Gly, Leu, Tyr, Phe and Pro). An enzyme activity unit (U_cbz) was defined as the amount of protease that releases 1.0 µM of p-nitrophenolate per min in the assay conditions.

Finally, the analysis of araujiain secondary structure in Tris-HCl buffer (pH 8), in ethylene glycol and DMF was carried out by Fourier transform infrared (FTIR) spectroscopy. The relative amount of different components from the band amide I of araujiain was determined by means of the second-derivative analysis

According to Figure 1, caseinolytic activity profiles of araujiain showed the highest retention (Ucas/mg of protein) in DMF and ethanol, but these values were 76% higher in the former than in the latter, after 4 hr at 40ºC. It is important to point out that caseinolytic activity retention of araujiain in 0.1 M Tris-HCl buffer (pH 8) was 2.1 Ucas/mg of protein. This value was 19 times lower than that obtained in DMF. 

At temperatures higher than 40ºC, the residual caseinolytic activity of araujiain diminished. The same behaviour was observed in buffer Tris-HCl although those organic solvents diminished the autolysis degree.

When araujiain substrates preferences in buffer Tris-HCl, ethylene glycol and DMF were studied, different patterns were observed (Table 1). A previous study demonstrated that the hydrophobic and steric factors of the side chains of the amino acids reduced the esterase activity of the araujiain in buffer while the electronic interactions were very important in the increase of that activity. When water was substituted by an organic solvent, these effects did not permit to derive generalizations on the araujiain preferences. Nevertheless, the low affinity of araujiain for the Pro, Phe and Tyr derivatives in DMF might be influenced by the esteric effect of the side chains of these amino acids, which reduced the esterase activity of the enzyme.

We hypothesized that the changes in araujiain stability and preferences could be related to the conformational changes of the enzyme in each medium. 

According to FTIR spectroscopy studies, the maximum of amide I band (which is known to be sensitive to protein secondary structure and conformational changes) of araujiain in buffer Tris-HCl, in ethylene glycol and DMF was observed at 1653 ± 2 cm^-1 (random structure) (Figure 2). Moreover, araujiain exhibits a lower α-helical character and greater β-sheet folding in buffer than in ethylene glycol or DMF.

In addition, the diminution of the β-sheet band to 1689 cm^-1 along with the increase of a β-sheet band at 1698 cm^-1, which corresponds to intermolecular bonds, indicate the formation of enzymatic aggregates when araujiain was dissolved in buffer Tris-HCl (pH 8).

A secondary structure with high α-helical character is likely to be the cause of the high hydrolytic and esterolytic activities of araujiain in DMF, in relation to the buffer. On the contrary, no correlation was observed between the activities of araujiain in ethylene glycol and the secondary structure.

Strictly, the correlation between enzyme structure and catalytic activity requires direct measurement of active-site structure and the effect of the reaction medium on the transition state. Nevertheless, according to FTIR spectra, it is clear that the non covalent forces that maintain the native secondary and tertiary structures of enzymes were modified when araujiain was suspended in some organic media with low water content.

These facts allowed to verify that araujiain suspended in an organic solvent such as DMF (with low water content) changed its secondary structure and its substrate preferences and adopted a native-like conformation but with higher stability, very good hydrolytic potential and higher esterolytic activity than in an aqueous medium.