Safflower seeds were supplied from National Institute of Agricultural Research of Tunisia. Seeds were imbibed overnight in water, and then placed to germinate at 27 °C in darkness with a water supply. The 5-d grown seedlings were harvested and kept in −20 °C until use. The cotyledons were homogenised in 4 volumes (w/v) of 0.1 M acetate buffer (pH = 6) containing 1 mM PMSF, 5 mM CaCl₂, followed by stirring slowly for 1 h at 4 °C. The homogenate was centrifuged 15 min at 10 000 g at 4 °C. The supernatant was recovered and kept at −20 °C until use.

A crude extract was prepared from 20 g of 5-day-old cotyledons as mentioned above. The extract was fractionated by gentle addition of ammonium sulphate up to 20 or 60% concentration of saturation, followed by centrifugation during 15 min at 10 000 g at 4 °C. The fraction 20–60% was recovered, dissolved and dialyzed against 50 mM acetate buffer (pH = 6) containing 5 mM CaCl₂.

Affinity chromatography using β-cyclodextrin Sepharose 6B prepared according to the method of Vretblad was performed [15]. To the crude extract 5% ammonium sulphate was added [7] and the solution was poured onto the column (1.6×40cm, 40 ml) pre-equilibrated in 50 mM acetate buffer (pH = 6) containing 5 mM CaCl₂. After the column was washed with 50 mM acetate buffer (pH = 6) containing 25 mM CaCl₂ and 0.5 M NaCl, the elution was effected by 8 mg/ml of β-cyclodextrin in the washing buffer. The active fraction was dialyzed against 50 mM acetate buffer (pH = 6) containing 5 mM CaCl₂ and was concentrated by lyophilisation. All purification steps were done at 4 °C.

The hydrolytic activity for soluble starch was measured in the standard reaction mixture consisting of 400 μl of 0.5% soluble starch prepared in 0.1 M of sodium acetate buffer (pH = 6.0) containing 5 mM CaCl₂ and 100 μl dilute enzyme with acetate buffer. The reaction was started by adding substrate. The reducing sugar produced in 10 min at 55 °C was measured by DNS (3,5-dinitrosalicylate) method at 540 nm by using glucose as standard reducing sugar. One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of reduced sugar per minute under these reaction conditions.

2.5.1 Molecular mass determination

2.5.2 Amylase activity staining

The effects of pH on soluble starch hydrolytic activity were examined with standard essay conditions, at pH values from 3.0 to 10.0. The pH optimum of purified enzyme was measured by using two different buffers for two different ranges, 0.05 M acetate buffer (pH 3–6.5) and 0.05 M Tris–HCl (pH 7–11). For pH stability, enzyme was kept at 4 °C for 24 h in buffer at different pH values.

The effects of temperature on soluble starch hydrolytic activity were examined with standard essay conditions, but changing the incubation during 10 min in sodium acetate buffer (pH = 6.0) from 4 to 90 °C. For studying the thermal stability, enzyme was kept at variable temperature for 30 min and residual activity was measured by standard essay.

Purified amylases were incubated with 5 mM and 10 mM solution of salts of metal ions (chlorides of Ca²⁺, Ba²⁺, Mg²⁺, Cu²⁺, Zn²⁺ and Zn²⁺) and their activities were determined. The enzyme activities without metal ions were taken as 100% and relative activities determined in the presence of metal ions were calculated.

In this study it was found that α-amylase from germinating safflower seeds showed maximum activity after 5 days of growth and then declined rapidly (Fig. 1). From this information, we decided to purify this enzyme activity 5 days following seed imbibitions. The existence of amylase activity in oleaginous seeds testifies the existence of the glucidic reserves, particularly starch. The origin of this starch is to be checked. Does this starch east exist in dry seed or is it synthesized during germination starting from other precursors, particularly the breakdown products of the lipids storage? Earlier work showed that the oleaginous seeds accumulate starch at the beginning of their germination [19,20]. Thus, it was shown that the content of starch in endosperm tissue of castor seed (Ricinus communis) increases from 0.5 to 1.1 mg per seed in 5-day-old seedlings [21]. Recently, another study showed, by proportioning and electronic microscopy, that the cotyledons of yellow lupine seeds (Lupinus luteus L.), which are regarded as leguminous seeds rich in lipids, accumulate starch and that the content of starch increases from 8 to 22 mg/g of dry matter in 4-day-old seedlings [22].

A summary of the purification procedure that was used for this cotyledonary α-amylase is presented in (Table 1). We have obtained 0.14 mg of purified α-amylase. Crude extract for 20 g of safflower cotyledons for 5-day-old seedlings of germination was fractionated by ammonium sulfate precipitation and separated into two fractions (0–20% and 20–60%). More than 95% of the activity was obtained in the 20–60% fraction. Ammonium sulphate fraction 20–60% was loaded onto an affinity chromatography column. After elution by 8 mg/ml cyclodextrin solution, amylase activity and proteins were found in one peak (Fig. 2). Safflower amylase was purified more than 131.52 folds with an 81.25% activity yield and its specific activity equaled 238 U/mg of protein (Table 1). The elution pattern of the Sepharose 6B-β-cyclodextrin column chromatography showed one peak of protein and of amylase activity (Fig. 2). The affinity chromatography technique used in this study has also been used successfully for amylases purified from Vigna mungo cotyledons [23,24], from Arabidopsis thaliana leaves [25], and from roots and cotyledons of Pisum sativum [26]. These results are in accordance with similar purification procedures used by others [23,25]. It can be observed that the enzymatic activity was eluted in one peak, which coincided with the peak of protein. Fractions of this peak (46–49) were collected and concentrated.

This purified cotyledonary α-amylase from 5-day-old safflower seedlings appeared to be present as only one isoform in the zymogram of α-amylase and protein band pattern of the purified enzyme (Fig. 3). Similarly Vigna mungo cotyledons from 4-day-old seedlings had only one α-amylase that exists mainly as a monomer that partially aggregates to form dimer, trimer and further multimers [23]. In contrast, cotyledonary amylases preparations obtained from Pisum sativum seedlings exhibit from two [27] to seven [28] α-amylase isozymes.

The molecular mass of this purified cotyledonary α-amylase was found by SDS-PAGE to be 35 kDa (Fig. 4). This value is very close to that obtained for the cotyledonary α-amylase purified from pearl millet α-1-amylase (31 kDa) [29], from Vigna mungo (43 kDa) [23], Azuki bean Amy1 (47 kDa) and Amy2 (44 kDa) [7], and Vigna Ungularis (45 kDa) [30] (Table 2).

The effect of pH on this enzyme activity was examined at constant temperature using sodium acetate and Tris–HCl buffers. As shown in (Fig. 5), purified safflower α-amylase showed an optimum pH value of 6.0. This optimum pH value is consistent with data reported for other amylases (Malted wheat α-1, Malted barley α-1, Malted finger millet α-2 [31,32]) and it is close to the optimum pH of azuki bean amylases: VAAmy1 (5.3) and VAAmy2 (5.2) (Table 2).

The range of pH stability, where the presently purified amylase conserved more than 60% of original activity after 24 h of incubation at 4 °C, was from 4.0 to 9.0 (Fig. 6). At values higher than 9, however, enzymatic activity dropped sharply to 50% of maximal activity.

The optimum temperature for safflower α-amylase activity is 55 °C (Fig. 7). This value is consistent with data reported for other plant amylases (wheat α-1 (55 °C), pearl millet α-1 (55 °C) [29]) and smaller than that for azuki bean amylase (70 °C) [7]. This enzyme was not stable up to 70 °C. For example, the enzyme lost 50% of its activity after 10 min incubation at 70 °C (Fig. 8), and was completely denatured at 100 °C.

Various metal ions such as Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺ and Cu²⁺, at 5 and 10 mM concentration were tested for amylase activation/inhibition. The results are given in Table 3. Ca²⁺ was found to have both activating and stabilizing effects as indicated by increased activity whereas Mg²⁺ had negligible effect on amylase activity. However, the enzyme inactivation in the presence of Zn²⁺ and Cu²⁺ was found to be partial. In contrast, Fe²⁺ completely inactivated this amylase. The inactivation by this metal may be due to its binding to either catalytic residues or by deplacing the Ca²⁺ from the substrate binding site of the enzyme. The role of Ca²⁺ in maintaining the stability and structure of the α-amylase is well documented [33].

Enhancement of amylase activity by Ca²⁺ ions is based on its ability to interact with negatively charged amino acid residues such as aspartic and glutamic acids, which resulted in stabilization as well as maintenance of enzyme conformation [34]. In addition, calcium is known to have a role in substrate binding [35]. It has also been documented that binding of Ca²⁺ to amylase is preferred to other cations such as Mg²⁺ [36].

To our knowledge, this is the first oleaginous α-amylase that has been purified. This purification opens many horizons to us to comprehend the germination metabolisms of these types of seeds, the physiological significance of which calls for study in future works.