Determination the Optimum Conditions of the Activity and Stability of Lipase Extracted from Sunflower Germinated Seeds

The present study was conducted to determine the optimum conditions required for lipase enzyme activity extracted from germinated sunflower seeds, including temperature, pH, agitation, time of incubation, enzyme concentration, substrate type, and concentrations of mineral salts and EDTA. Optimum pH, temperature and time of incubation required for lipase stability were also determined. The results showed that the optimum lipase activity (3.251U/ml) was foun t and pH 7 after 20 minutes of incubation when using 1 ml lipase enzyme with 0.02 ml of CaCl2 (10 mM) at 100 rpm of agitation and in the presence of olive oil as the substrate for enzyme reaction. EDTA appeared to have inhibitory effects, while Ca +2 and Mg +2 have stimulatory effects on lipase activity. The values of lipase activity, total activity, and specific activity measured under optimum conditions were increased by 36.99%, 36.95%, and 38.21% over control, respectively. The enzyme showed st bility t temper ture r nge between to 5  ̊ , pH between 7 to 8, and time of incubation between 10 to 40 minutes. These results suggest that lipase enzyme extracted from germinated sunflower seeds have stability that depends on pH, temperature, and incubation period, which enables it to be used in different industries.


Introduction
Lipases (triacylglycerol ester hydrolase, EC3.1.1.3) are enzymes that catalyze the total or partial hydrolysis of fats and oils, releasing glycerol and free fatty acids [1]. The purified enzyme is used in a wide range of industries, such as food industries [2], detergent industries [3], and biodiesel production from wastes and non-edible vegetables oil, with the aims of reducing the high cost of biodiesel production and other uses [4][5][6][7].
Lipases activity is the total ability of lipases to hydrolyze lipids and fats and produce fatty acids and glycerol. The activity was found to be affected by various factors, such as temperature, pH, agitation, time of incubation, enzyme concentration, type of substrate, minerals and EDTA. Lipase stability is also affected by time of incubation, pH and temperature. The optimum of these factors was found to be varied among the enzymes and their varieties in the same plant species [8,9]. Therefore, it has become necessary to determine the optimum aforementioned factors in order to obtain the higher specific activity and stability of the tested enzyme.
In an earlier work [10], lipase was extracted from sunflower seeds by Tris-HCl buffer at 72 h after germination. Purification of the enzyme was also made; however the characteristics of the enzyme were not investigated. Therefore, the present study was conducted to determine the optimum temperature, pH, agitation, time of incubation, enzyme concentration, type of substrate, minerals and EDTA which are required for maximum lipase activity. Incubation time, pH and temperature for maximum lipase stability were also determined.

Materials and Methods Lipase extraction and purification
Lipase was extracted from sunflower germinated seeds after 72 h of seeds germination and purified by procedure outlined by earlier work [10]. The enzyme was used to determine the optimum value of the following factors.

Effect of incubation time on lipase activity
The reaction mixture consisting of enzyme, oil emulsion substrate, and CaCl 2 was incubated at 5, 10, 20, 30, 40, 50, 60, 70, and 80 minutes in a shaker water bath, then the enzyme activity was measured to determine the optimal time for incubation [11].

Effect of pH on lipase activity
pH of the reaction mixture that could support maximal lipase activity was determined by adjusting the pH of reaction mixture to 4, 5, 6,7, 8, and 9 using different types of buffers, including Tris-HCl buffer (pH 8,9), phosphate buffer (pH 6, 7), and acetate buffer (pH 4, 5). Then, the lipase activity was measured.

Effect of incubation temperature on lipase activity
Optimal incubation temperature for maximal lipase activity was evaluated by incubating the reaction mixture at different temperatures of 25, 30, 35, 40, 45, n 5 , t en lip se tivity w s measured [11].

Effect of agitation on lipase activity
The effect of agitation on lipase activity was studied by the incubation of the mixture of reaction at 0, 25, 50, 75, 100, 125, and 150 rpm in shaker water bath. Lipase activity was determined at the end of the incubation period [11].

Effect the type of substrate (oils) on lipase activity
The effects of various types of substrates on lipase activity were tested. Sunflower oil, soy beans oil, coconut oil, and walnut oil were added to the mixture of reaction instead of olive oil and incubated in shaker water bath, then the activity of lipase was measured. The lipase activity from olive oil was used as a standard value for the tested oils [11].

Effects of mineral salts and EDTA on lipase activity
Salts of CaCl 2 , MgCl 2 and KCl as well as EDTA, at concentrations of 0, 5, 10, 15 and 20 mM, were added to the reaction mixture to test their effects on lipase activity. The activity was measured by spectrophotometer at 715 nm at the end of the incubation period [11].

Lipase activity with and without using optimal conditions
Lipase activity was measured using the optimal conditions obtained from the previous experiments (1 ml enzyme concentration, 2.5 ml olive oil emulsion, 0.02 ml CaCl 2 , 20 minutes incubation time, ˚ , pH t 7, and 100 rpm of agitation ). Conditions utilized by Rahman et al. [11] were used for comparison. Factors affecting lipase stability 1.
Effects of temperature on lipase stability e enzyme solution w s in ub te in w ter b t t temper tures between to 9 or minutes t t e en o e in ub tion perio , t e enzyme w s r pi ly oole t or min and tested by adding it to the reaction mixture (olive oil emulsion, buffer, and others). After the end of incubation period, the absorbance was read at 715 nm to determine the enzyme activity.

Effects of pH on lipase stability
The effects of pH on lipase stability was determined by incubating 1 ml of enzyme in various buffer solutions ranged from 3 to 9, including acetate buffer with pH 4 and 5, phosphate buffer with pH 6and 7, and Tris-HCl buffer with pH 8 and 9, in shaker water bath. Lipase activity was then measured [11]

Effects of time on lipase stability
Time is a very important factor that affects lipase stability. One ml of the enzyme was incubated in water bath for different times (0, 10, 20, 30, 40, 50, 60, 70, and 80 minutes). Then, the enzyme was tested by adding it to the reaction mixture (olive oil emulsion, buffer, and others). After the end of incubation period, the absorbance was read at 715 nm to determine the enzyme activity.

1-Effects of incubation periods (time) on lipase activity
The results showed that 20 minutes of incubation for the mixture of the enzyme and substrate in shaker water bath exerted the maximum activity (1.87 U/ml). The lipase enzyme remained active until 30 minutes then the activity declined sharply (Figure-1). The decline in enzyme activity after 30 minutes of incubation could be either due to the decrease in the substrate availability or the catabolizing repression of the enzyme.

Effects of pH on lipase activity
Lipase showed the highest activity (2.3 U/ml) at pH 7. This suggests that the lipases extracted from sunflower seeds prefer neutral pH conditions, giving the maximum activity when Tris-HCl buffer at pH 7 was used in the experiments (Figure-2). Several investigators reported that oil seeds of different plant species contain acid, alkaline, or even neutral lipases. Madhikar et al. [8] demonstrated that the enzyme activity in sunflower germinated seeds was increased with increasing pH, with maximum activity observed at pH 7. Sagiroglu and Arabaci [12] showed that the maximum activity of sunflower lipase was achieved at pH 7.5. Muto and Beevers [13] found that castor bean seeds have two types of lipases, acid lipase and alkaline lipase, with maximum activity at pH 5 and 9, respectively. Eze and Chilaka [14] suggested that white melon seeds contain acid lipase and alkaline lipase with optimum activity at pH 4.5 and 7.5, respectively. The optimum pH for lipase activity depends on the source of enzyme, the substrate used, and the components of the assay reaction [14].

3.
Effects of incubation temperature on lipase activity ip ses extr te rom sun lower germin te see s s owe m ximum tivity 9 ml t temper ture o igure-3). This result agrees with the study of Ezema [15] who found that the two types of lipase extracted from white melon optimum tivity t t er stu ies reporte that maximum activity of lipases extracted from white melon and soya bean germinated seeds was achieved at temperatures o n , respectively [14,16]. Different earlier studies showed that t e optimum temper ture or m ximum enzyme tivity w s , as in cellulose extracted from a local isolate of Pantoea spp. [17] and peroxidase extracted from Brassica oleracea Var. [18].

Effects of enzyme concentration on lipase activity
The results presented in Figure-4 shows that lipase activity was increased gradually with increasing the concentration of the enzyme, until it reached 2.36 U/ml at a concentration of 1.75 ml, then remained constant even when lipase concentration was increased to 2 ml. Also, the results showed that the activity was 2.31 U/ml at 1 ml, while it was 2.36 U/ml at 1.75 ml of enzyme. This implies that there is a little (0.05 U/ml ) increase in lipase activity when 1.75 ml of enzyme is used. However, this increase is not considered as economically valuable. Therefore, 1 ml of lipase concentration was the optimum concentration of enzyme that exerted the highest activity (2.31 U/ml). The increase of enzyme concentrations will increase the rate of reaction as more enzymes causes more colloid with substrate molecules [19], but further increasing lipase concentration has no further effect on the rate of hydrolysis as all enzyme molecules are saturated with substrate molecules [20].

5.
Effects of agitation on lipase activity The results presented in Figure-5 show that agitation at 100 rpm was quite sufficient to yield optimum lipases activity (2.31 U/ml), since the activity remained almost the same at 125 and 150 rpm. Agitation rate may affect the viability of the enzyme to the substrate during the reaction of lipase activity, while higher agitation rate may affect the structure of the enzyme [21].   Figure-6 shows that different types of oils used in the mixture of reaction caused different values of lipase activity. Olive oil exerted the highest value of activity (2.31 U/ml), followed by walnut oil (1.98 U/ml), while coconut oil had the lowest value (0.28 U/ml). It was reported that the activity of lipase increases or decreases according to the type and concentration of oil used [22]. The oil substrate may change the physical and chemical characters of lipase enzyme and higher oil concentration may not be suitable for lipase activity or even becomes inhibitory to lipase enzyme.

Effects of mineral salts and EDTA on lipase activity
The effects of mineral salt ions and EDTA on lipase activity were different according to the type and concentration of mineral salts and EDTA used (Figure-7). The results showed that CaCl 2 , MgCl 2 and KCl at 10 mM caused an increase in residual lipase activity, which reached 146.5%, 119.5%, and 109.8%, respectively, while EDTA at 10 mM caused a decrease in residual lipase activity, reaching 86.2%. Therefore, EDTA manifested significant inhibiting effects on lipase activity at a concentration of 10 mM, as well as at other concentrations. This inhibitory effect may be due to the effect of EDTA on the interaction between the enzyme and substrate (oil). The inhibitory effects of EDTA, which acts as chelating agent, on lipase are also reported in seeds of different plant species, such as African bean [23], castor bean [24], white melon [14], and mustard and rape [25]. The higher activity of lipases caused by Ca +2 is also reported by Madhikar et al. [8] who studied the lipases of sunflower germinated seeds, while Kermasha and Van de Voort [26] reported that Ca +2 has an inhibitory effect on lipases extracted from French bean. The presence of Ca +2 and Mg +2 increases the activity at low concentrations, while Hg +2 and EDTA decrease the activity of lipase extracted from Soybean seeds [16].

Compression of lipase activity and specific activity measured under optimum and non-optimum conditions
The results indicated that the optimum conditions for the specific lipase activity are quite different from conditions adopted by Rahman et al., [11]. Temperature, time of incubation, and agitation rate were found to be obviously decreased in the present optimum conditions, compared with in the conditions utilized by Rahman (Table-1). The other factors remained the same. The results also showed that lipase activity, total activity, and specific activity values measured by optimum conditions were increased by 36.99%, 36.95% and 38.21%, respectively, over values achieved by Rahman (Table-2). The enzyme concentration, the amount of olive oil, and the amount of CaCl 2 in both conditions were almost the same.

Factors effecting lipase stability 1. Effects of temperature on lipase stability
The results indicated that lipase extracted from sunflower germinated seeds was stable at a temper ture between to 5 ˚ , then the activity started to decline sharply, but still detectable below 5 ˚ , in i ting t t t e enzyme is thermo-stable (Figure-8).  Figure-9 indicates that lipase was stable at pH 7 -8 with activity values of 2.29 U/ml and 2.3 U/ml, respectively. This suggests that the enzyme was stable in neutral and weak alkaline solutions.

Effects of time on lipase stability
The residual activity of the tested lipase was found to be stable at time ranged 10 -40 minutes, then decreased gradually (Figure-10). This result suggests that lipase enzyme is less stable for a long period of time.

Conclusions
Based on the results, the optimum conditions for lipase extracted from sunflower seeds were determined and found to cause increased lipase activity, total activity, and specific activity by 36.99%, 36.95% and 38.21%, respectively, over the values obtained via the conditions used by Rahman et al. [11]. The enzyme showed stability at a temperature ranged between 30 and 5 ˚ , pH between 7 and 8, and time of incubation between 10 and 40 minutes. These results suggest that lipase enzyme extracted from germinated sunflower seeds has stability to pH, temperature, and incubation period, which enables its utilization in different industries.