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Introduction
By-products generated in the agricultural industry cause much environmental pollution and adverse health effects (Hamrouni et al., 2020; Leite et al., 2021). Waste resulting from olive oil extraction creates severe environmental problems with its highly polluting properties (Mantzavinos and Kalogerakis, 2005; Sarika et al., 2005). Olive waste contains biodegradable compounds and phytotoxic phenolic compounds. Phytotoxic wastes correspond to about 80 % of olive oil production. These phenols tend to change into condensed high molecular weight polymers, which are difficult to degrade on storage (Ayed et al., 2005). For these reasons, olive oil wastes may lead to acute odor problems and, more importantly, serious risks for water and soil quality (Mantzavinos and Kalogerakis, 2005; Sarika et al., 2005). Today, with the increasing awareness of environmental protection, the use of biomass has gained importance (Hamrouni et al., 2020). Olive oil waste contains simple and complex sugars, lipids, residual oil, proteins, and mineral elements, besides phytotoxic wastes. These compounds can be directly recovered by chemical extraction followed by purification (Fki et al., 2005; Papadimitriou et al., 2005). Olive oil waste can be used as the basic compound for fermentative production processes (Fenice et al., 2003; Angenent et al., 2004). Various agro-industrial wastes have been used for biotechnological purposes, especially for enzyme production (Mahanta et al., 2008). Lipases are one of those enzymes, which are commercially important since they catalyze the hydrolysis of long chain fatty acids to glycerol and fatty acids.
Most commercial lipases are produced by microorganisms (Babu and Rao, 2007; Treichel et al., 2010; Bharathi and Rajalakshmi, 2019; Adetunji and Olaniran, 2021). Especially since the lipase enzyme produced from lactic acid bacteria is considered safe, it is preferred in the food industry (Meyers et al., 1996; Liu et al., 2001; Lopes et al., 2002; Couto and Sanroman, 2006; Ramakrishnan et al., 2013; 2015; 2016; Sukohidayat et al., 2018; Dellali et al., 2020; Acu et al., 2021).
In our study, it was aimed to produce, partially purify and characterize the lipase enzyme from E. faecium E68 strain in olive oil waste.
Material and methods
Bacteria
In the study, Enterococcus faecium obtained from Eskişehir Technical University microbiology unit was used. E. faecium E68 was inoculated in M17 broth and incubated at 37 °C for 24 h under 10 % CO2 conditions. Growing cultures from M17 broth were inoculated on M17 agar and incubated at 37 °C for 24 h under 10 % CO2 conditions. The morphological features of the colonies formed after incubation were examined. Then, the purity of the cultures was checked by microscopic examination by Gram staining.
Olive oil waste
Olive oil waste with dry matter content of 0.82 g, nitrogen content of 1.593 % and oil content of 0.036 % was obtained from olive oil production facilities and used in the studies.
Lipase production
E. faecium E68 was inoculated onto M17 agar and incubated at 37 °C for 48 h. It was then inoculated as a single colony into M17 broth and incubated at 37 °C for 24 h. Culture was set to an Optical Density (OD) of 1 at 600 nm in the spectrophotometer. Then, 1 % of the culture was inoculated into the lipase determination medium.
The study was carried out in 2 parts. In the first part, without adding olive oil waste to the medium, and in the second part, by adding 10 % olive oil waste to the medium.
For enzyme production, 500 mL of lipase assay medium was prepared, added with 5 % peptone as nitrogen source, 3 % glucose as carbon source and other components (0.1g/L CaSO4, 0.5g/L KH2PO4, 0.1g/L MgSO4 x 7H2O, 1 % tributrine) and the pH adjusted to 6.5-7. It was incubated for 48 h in a 37°C shaking oven (120 rpm) under 10 % CO2 conditions. After incubation, it was centrifuged at 9,798 x g for 30 min at 4 °C. The cooled acetone was mixed with the obtained filtrate at a 1:5 ratio, kept at 4 °C for 24 h, and centrifuged at 9,798xg, 4 °C for 15 min.
In the application where olive oil waste is used, the same processes were applied by adding 10 % olive waste to the lipase medium (0.1 g/L CaSO4, 0.5 g/L KH2PO4, 0.1 g/L MgSO4 x 7H2O, 1 % tributrine).
Then, 3 mL of the enzyme was placed into the dialysis tube (Sigma PURX12015), which in turn was placed in Tris HCl buffer. Tris HCl buffer was changed every 24 h. After 48 h, partially purified enzyme was obtained. Partially purified enzymes were used in the experiments. Experiments were carried out in two replicas each.
Assay of lipase activity
Para-nitrophenyl palmitate (p -NPP) method, which is a spectrophotometric method, was used for lipase activity determination (Arora, 2013). This method determined lipase activity by measuring p-NPP at a wavelength of 405 nm in a spectrophotometer. One unit (U) of lipase activity is the amount of enzyme that releases 1 µmol p-nitrophenol per unit time (min).
Determination of the molecular weight of lipase
The protein amount of the lipase enzyme was determined by the Bradford method (Bradford, 1976) in a spectrophotometer at 590 nm using Coomassie Brilliant Blue G-250 dye. The molecular weight of the enzyme was determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) according to the method of Laemmli (1970).
Determination of Factors Affecting Partially Purified Ex-tracellular Enzyme Activity
Effect of temperature and pH on enzyme activity
To determine the effect of temperature on partially purified extracellular enzyme activity, the enzyme was incubated for 1 h at 5 oC, 20 oC, 30 oC, 37 oC, 40 oC, 45 oC, 55 oC and 65 oC in 50 mM sodium phosphate buffer (pH 7).
To determine temperature stability, lipase enzyme was placed in 50 mM sodium phosphate buffer (pH 7) and incubated at 5 oC, 20 oC, 30 oC, 45 oC and 55 oC for 5 min, 1 h, 4 h, 24 h and 48 h. After cooling, the remaining enzyme activity was measured (Esteban-Torres et al., 2015).
The effect of pH on lipase activity was determined by keeping the enzyme in buffers prepared between pH 3-11. Acetic acid-sodium acetate buffer was used for pH 3-5, sodi-um phosphate buffer for pH 6, Tris-HCl buffer for pH 7-8, and glycine NaOH buffer for pH 9 (Esteban-Torres et al., 2015).
In order to determine the enzyme pH resistance, 200 µL of buffers at different pHs were placed in the microtubes. On top of it, 200 µL of enzyme were added and incubated at 45 °C for 2 and 3.5 h. The remaining enzyme activity was determined (Esteban-Torres et al., 2015).
Effect of some surfactants and cations on enzyme activity
To determine the effect of some cations, surfactants, and solutions on the enzyme activity, MnCl2, CuCl2, MgCl2, KCl, NiCl2, CaCl2, HgCl2, and ZnCl2 of 1 mM were added, and the enzyme activity determined at 405 nm in a spectrophotometer.
The effect of urea, EDTA, SDS, tween 20, tween 80, and triton X-100 on enzyme activity was determined by adding 1 µL to the medium (Esteban-Torres et al., 2015, Ramakrishnan, et al., 2016).
Effect of salt on enzyme activity
To determine the effect of sodium chloride (NaCl), it was added to the buffer at concentrations of 0 %, 1 %, 5 %, 10 %, 15 %, 20 %, 25 % (w/v) and the enzyme activity determined in spectrophotometer at 405 nm (Esteban Torres et al., 2015).
Result and discussion
In recent years, producing useful substances from waste materials has been of great importance. Thus, products with economic importance can also be obtained while preventing environmental pollution. In the study, it was determined that olive waste could be used in the nutrient medium and, in this way, a product of high economic importance can be obtained.
The extracellular lipase enzyme obtained without using olive oil waste and using 10 % olive oil was partially purified and used in the tests. The wet and dry weights and protein contents of the obtained lipase enzymes are given in Table 1.
The protein amounts in the extracellular enzymes were found to be 0.315 mg/mL for the lipase enzyme produced in the medium, while it was 0.691 mg/mL for the partially purified enzyme produced in olive oil waste.
As a result of E. faecium E68 SDS-PAGE analysis, the molecular weight of lipase enzymes was determined at around 19-20 kDa (Figure 1).
The effects of temperature, pH, cations, and surfactants on the enzyme activity were determined.
The partially purified extracellular enzyme showed high lipolytic activity between 5 °C and 65 °C. The highest activity of the enzyme produced in the lipase medium was between 5 - 20 oC (Figure 2). Partially purified extracellular enzyme activity obtained with E. faecium E68 in olive waste was highest at 45 oC (Figure 2). Temperatures above 45 oC caused a decrease in enzyme activity (Figure 2). It has been reported that the partially purified extracellular enzyme obtained from E. durans E114 shows maximum activity at 30 - 45 oC (Acu et al., 2021). Maximum activity in the E. faecium lipase enzyme was observed at 40 °C (Ramakrishnan et al., 2016). Dellali et al. (2020) reported that the optimum lipase activity of E. faecium strains was 30-40 oC.
Lipase enzyme production was performed with E. faecium E68 in lipase medium containing 10 % olive oil waste (pH 6.5) after 48 h of incubation at 120 rpm at 37 oC.
Higher enzyme activity was obtained in the extracellular enzyme produced by E. faecium E68 in the fattening medium where olive waste was used. Lipid sources, such as natural oils have been shown to stimulate lipase production. Olive oil is one of the best inducers of lipase production (Zarevúcka, 2012). The presence of a certain amount of olive oil in olive oil waste stimulated lipase production. The activity of intracellular and extracellular lipases increases with increasing lipid concentration (Zarevúcka, 2012). The reason for the higher activity of the lipase enzyme produced using olive oil waste may be related to the increased lipid concentration.
Partially purified enzyme activity produced in olive waste decreased at 20 oC and remained stable at 55 oC, although it was below the optimum activity. The lipase enzyme produced in the lipase medium maintained its activity at 20 oC (Figure 3). The temperature tolerance of the extracellular enzyme remained quite stable after an incubation period of 24-48 h at 30-45 °C. Ramakrishnan et al. (2016) reported that the E. faecium lipase enzyme activity is stable between 30-70 oC. Researchers have reported that the enzyme activity is stable at 80-100 oC, and that enzyme activity does not remain after 100oC. Esteban Torres et al. (2015) observed that the maximum activity of the L. plantarum esterase enzyme is at 40 oC. They reported that the enzyme showed only 40 % of its activity at 5 oC, and, after 10 h of incubation at 55 oC and 65 oC, 40 % of the activity remained. Francisco et al. (2019) reported that the decrease in enzyme activity with temperature is associated with the change in its three-dimensional structure. It has been found that the alpha helix decreases at temperatures above 50 oC. At temperatures above 70 oC, the beta sheet increases while maintaining a low alpha helix. Opening the protein results in permanent inactivation and denaturation (Ismail et al., 2021).
The highest activity of the lipase enzyme, produced in olive waste and partially purified, was obtained at pH 10 (Figure 4). Partially purified enzymes were alkaline in nature. The optimum pH was found to be 10. While the lowest activity is obtained at pH 6, the enzyme has higher activity at pH 3. Acidic pH activity has been observed for a lipase from E. du-rans 27 isolated from fish processing waste. Lipase from ED-27 showed optimal activity at pH 4.6 and at temperature 30 °C (Ramakrishnan et al., 2015). A highly alkaline extracellular lipase that exhibits maximum hydrolytic activity at pH 10.8 has been reported from E. faecium MTCC5695 (Ramakrishnan et al., 2016). Dellali et al. (2020) reported that the optimum activity of the enzyme produced by E. faecium strains is between pH 6 and 9.
The pH stability of the extracellular enzymes obtained from E. faecium E68 was determined by incubating them at different pH values for 2 h and 3.5 h at 45 oC. The lipase produced in olive oil waste by E. faecium E68 remained significantly stable after 2 h and 3.5 h at pH 3 (respectively % 77,36 and % 70,79). However, the lipase activity produced in the lipase production medium was lower at pH 3. Similar acidic pH activity has been observed for a lipase from E. durans NCIM5427 from fish waste isolated from slaughterhouse waste (Ramakrishnan et al., 2015). For the enzyme produced in the lipase production medium, the activity loss was higher after 2 h and 3.5 h at pH 3. The highest loss of activity was observed at pH 5. Enzymes remained stable at alkaline pH for 2 h and 3.5 h of standing (Figure 5).
Lipolytic isoenzymes from a thermophilic Bacillus sp. have also been observed. It showed optimum activity at pH 8.5 and was reported to be very stable at pH 6.0 - 8.0 (Nawani and Kaur, 2007).
The effects of some ions and additives on the enzyme activity are given in Table 2. One mM Ca2+, Mn2+, Cu2+, Ni2+, Zn2+, Mg2+ and K+ ions significantly increased the lipase activities produced in olive oil wastes. Contrary to our findings, Ramakrishnan et al. (2015) reported that it significantly reduced Ca2+ and Mg2+ lipase activity. Mercury (Hg2+) led to a strong decrease in lipase activity. In the lipase enzyme produced in the lipase environment, metals other than Mg decreased the enzyme activity (Table 2). EDTA, which can affect the interface region between substrate and lipase, increased enzyme activity, however, some studies have reported that it reduces activity (Sztajer et al., 1992). The activity of lipase Lp_3562 was strongly inhibited by Hg2+, Cu2+ and SDS (Esteban-Torres et al., 2014a). Urea, Hg+2, Mn+2, Cu+2, Ni+2, Zn+2 and SDS, inhibited the activity of the enzyme produced in the lipase medium. Similarly, Dellali et al. (2020) reported that although the effect of metal and additive ions on esterase activity varies from bacteria to bacteria, they inhibit SDS, NaN3, CuCl2, EDTA, AgNO3 and HgCl2 enzyme activity.
Tabla 2 Efecto de aditivos sobre la actividad lipolítica de E. faecium E 68.
| Additives | Relative activity (%) | |
|---|---|---|
| Medium | Olive oil waste | |
| Control | 100 | 100 |
| HgCl2 | 41.7 | 48.9 |
| CaCl2 | 104.7 | 79.3 |
| MnCl2 | 112.8 | 88.2 |
| CuCl2 | 133.9 | 85.8 |
| NiCl2 | 110.0 | 86.1 |
| KCl | 130.8 | 90.9 |
| ZnCl2 | 105.2 | 89.2 |
| MgCl2 | 150.9 | 123.3 |
| EDTA | 101.4 | 108.4 |
| Urea | 128.7 | 92.8 |
| SDS | 87.3 | 91.2 |
| TritonX-100 | 81.5 | 120.5 |
| Tween20 | 104.6 | 93.5 |
| Tween80 | 105.4 | 128.5 |
While Triton X-100 and Tween 80 of surfactants increased enzyme activity, Tween 20 and SDS decreased enzyme activity.
This enzyme has high salt resistance and shows halophilic properties, an important feature in the preparation of foods. Even an increase in enzyme activity was observed (Figure 6). The salt resistance of this enzyme is high, and it showed halophilic properties. This feature is important for the preparation of foods.
The lipase enzyme produced in olive waste is promising in the food industry due to its resistance to 55 oC for 48 h, not losing its activity at low temperatures, and its halophilic properties. The use of these lipases is important as they can provide some advantages in food production.
The activity of lipase enzyme produced in olive oil waste was higher. The reason for this may be the presence of a small amount of olive oil residue in it. Olive oil has a significant effect for increasing lipase activity. It has been reported that the most suitable inducer in lipase production is olive oil. This has been associated with high levels of unsaturated grade free fatty acids, particularly oleic acid, in oil (Amenaghawon et al., 2022). This has been shown to facilitate cell growth and consequently increase both intracellular and extracellular lipase activity (Suci et al., 2018). A similar observation was reported by Brozzoli ve ark. (2009) and Rajendran and Thangavelu (2012).
Conclusion
The results revealed that natural substrate such as olive oil waste has good inducing properties for lipase synthesis. Therefore, it may be beneficial to use olive oil waste as a cost-effective source for lipase production. The relative stability of E. faecium E68 lipase at high temperatures may make it usable for biotechnological processes, as enzymes that can withstand high temperatures longer, attract the attention of industries. It is important that the lipase enzyme produced by E. faecium E68 has high activity at 45 oC and pH 10, as well as showing activity in acidic conditions such as pH 3. It is promising in the food industry with its resistance to 55 oC for 48 h, its effectiveness at low temperatures and its halophilic feature. Olive oil can be an important substrate for waste lipase production. Thus, environmental pollution can be prevented, and a biotechnological product is also obtained.
