Isolation, Purification, and Characterization of Pecan Nut Lipase and Studying its Affinity towards Pomegranate Extracts and 1, 4- Diacetoxybenzene

This study included partial purification of the lipase enzyme from the karnel of the pecan nut. After applying ion exchange chromatography technique using CMCellulose, two lipase isoenzymes were observed with specific activity values of 2.66 and 1.7 units/mg protein. The highest activity of both isoenzymes appeared at the optimum pH values of 8 and 6 and at temperatures of 40 and 50 °C, respectively. A pure single band was obtained by using electrophoresis technique and it was found that the approximate molecular weight was 42 kDa for the two isoenzymes. The study dealt with the preparation of the diester compound 1,4-diacetoxybenzene, which was diagnosed by GC-MS and 1 H NMR spectrometry. When studying the inhibition mode, the above compound showed a competitive inhibition of the lipase, as the value of Km increased from 0.013 to 2.857 mM, and Vmax was 0.25 enzymatic units / ml / min. Inhibition constant Ki was calculated and its value was 0.0319 mM. Pomegranate peel extracts with chloroform and ethyl acetate revealed an activating effect on lipase. This research aims to detect the presence of lipase in the pecan nut karnel and purify it by biochemical methods to know its properties and molecular weight. Then we attempted to inhibit it with a laboratory-prepared compound that has a structure similar to the basic substance on which the enzyme works.


Introduction
The pecan [Carya illinoinensis (Wangenh.) K. Koch] is a vegetable that belongs to Juglandaceae family. The pecan tree is a big tree that grows to be about 20 to 30 meters tall, with a crown diameter of 12 to 20 meters and a trunk circumference of 2 meters in ideal conditions. Pecan is a deciduous tree with alternate, pinnate leaves that grow up to 45 centimeters in length and are made up of nine to fifteen leaflets [1][2]. Pecans are notable among foods for their medical benefits, as they contain phytosterols, mono-and polyunsaturated fatty acids, phenolic compounds, and essential nutrients that decrease the severity of diseases [3]. According to Eagappan & Sasikumar [4], daily intake of nuts has been linked to several health benefits, including protection from oxidative damage, reduction of LDL-C, and prevention of chronic diseases, in addition to their antidiabetic and anticancer properties. They can help to reduce inflammation by decreasing the amount of inflammatory mediator molecules produced. Also, they can counterbalance the pro-inflammatory effects of a diet high in saturated fatty acids, which are commonly overconsumed in the Western diet [5]. Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are monomeric proteins with molecular weights ranging from 19 to 60 kDa that are ubiquitous and important in biology and industry. Natural lipases hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids, and glycerol. Esterases, as well as lipases, can hydrolyze carboxylic esters bonds [6][7]. Lipase enzymes have the unique ability to hydrolyze the ester bonds of poorly soluble substrates at the interaction between substrate and water. It has attracted a lot of consideration in latest years because of its assorted biotechnological applications [8][9]. Lipase is stable and can be obtained from animals, plants, and various microorganisms. Nature's catalysts, not only microbial lipases, are widely used commercially [10]. The lipase activity relies on many factors like surface area, extreme mild conditions, and pH. Purification entails not only isolating lipases from contaminants and sources, but also improving their stability, activity, and shelf life. Only when lipases have been purified to homogeneity can various studies on their structural orientation be conducted [11]. Because of their vast properties and ease of mass production, lipases among the most important enzymes. Lipases are useful for synthesis and have a wide range of applications in food preparation, fats and oil processing, paper manufacturing, and pharmaceuticals and cosmetics production [12]. Pomegranate peel is food waste obtained during the production of pomegranate juice and is unfit to eat. The peels make up about 40% of the total fruit weight. As a result, processing waste such as peel has emerged as an ideal substrate for extracting valuable bioactive compounds [13]. Phytochemicals have been found in various parts of the pomegranate fruit, including the juice, seeds, and peels, according to previous research [14]. Many polyphenolic compounds, such as gallic acid, caffeic acid, and coumarin, were separated from pomegranate peels by HPLC [15]. Solvent extraction is a method for isolating plant antioxidants [16]. Due to its various chemical and polar properties, the phenolic content of various plant materials is dependent on the nature of the solvent used [17]. Many studies have discovered that the phenolic content of plants varies depending on the polarity of the solvent [18]. Pomegranate peel extracts have recently drawn attention due to their potential as natural food preservatives [19].

Plant material and Chemical
In this paper, pecan nut was used, which was seasonally obtained from the local markets in the city of Ankara in November, 2020, and was classified in the Faculty of Education, Department of Life Sciences (C. illinoinensis) (Picture 1). The process of isolating the shell from the pulp was conducted directly for subsequent experiments on the pulp. All chemicals used were of analytical grade and obtained from Sigma and Molecule Chemical Company.

Protein Determination
Lowery method was used to estimate the concentration of protein by using bovine serum albumin as a standard [20].

Preparation of crude Lipase extraction
Fifty gram of pecan nut karnel was mixed with cold distilled water in a ratio of 1:4 weight: volume and crushed for ten minutes using the Blender machine. Then, it was frozen by adding liquefied nitrogen and allowed to melt at room temperature and the process was repeated three times. The mixture was filtered through several layers of gauze and the extract was separated by a refrigerated centrifuge for 15 minutes at a speed of 10,000 xg. The volume of the filtrate was measured and was found to be 70 ml.

Lipase assay
The activity of lipase enzyme was estimated using the method of Winkler and Stuckman [21]. The method includes the decomposition of the substrate p-nitrophenyl acetate, a reaction that is stimulated by the lipase enzyme. The method is based on measuring the absorbance of the para-nitrophenol at the wavelength of 410 nm.

Purification of Lipase
The procedures for lipase purification from pecan involved the following steps: 2.5. 1 Step 1: Ammonium sulfate precipitation Solid ammonium sulfate was gradually added to the crude extract, with a degree of saturation ranging 0-70%, taking into account the continuous stirring for one hour by the magnetic stirrer at 4 °C. The solution was left in the refrigerator for 24 hours to settle. The precipitate was separated with a refrigerated centrifuge for 20 min at 10,000 xg. The filtrate was discarded and then dissolved with the lowest possible amount of buffer phosphate buffer pH 7.1 at a concentration of 10 mM [22].

Dialysis
Dialysis was achieved by putting enzyme solution in a dialysis tube against the buffer on a stirrer at 4˚C with six-time buffer changes. This step was performed to remove the excess of ammonium sulfate ions.

Step 2: CM-cellulose column chromatography
The enzyme solution was placed onto CM-cellulose column (2×25 cm) before being equilibrated with buffer. The purification was accomplished by stepwise elution with increasing concentration of buffer at a flow rate of 1 ml/ min. Fractions showing tyrosinase activity were collected after monitoring at intervals of an individual fraction (3ml) and then lyophilized [23].

Characterization of Lipase
By using 1.5 mM of P-nitrophenyl acetate as substrate, the determination of lipase activity was carried out at temperatures between 20 and 50 °C. The optimum pH was measured by using 10 mM potassium phosphate buffer (pH of 6-8).

Determination of Molecular weight
The molecular mass of the lipase purified after ion-exchange chromatography was estimated by using polyacrylamide of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by following the Laemmli method [24]. In bathwater, the protein solution was heated for 5 minutes at 100 °C. The stacking gel was run at a voltage supply of 70 V for about 45 minutes, and the separating gel was run at a voltage supply of 100 V for about 90 minutes. The Coomassie Blue staining technique was used to visualize the protein bands, and the molecular weight was calculated using eight proteins of known molecular weights that ranged 14-116 kDa.

Preparation of 1,4-diacetoxybenzene
In a round bottomed flask, concentrated sulphuric acid (1 drop) was added to mixture of hydroquinone (2.2 g, 20 mmol) and acetic anhydride (4 ml, 40.4 mmol). Then, at room temperature, the reaction mixture was stirred until the hydroquinone was dissolved (exothermic reaction). The reaction was cooled and poured unto ice-water (30 ml), to bring about the product to precipitate [25][26]. The precipitated product was collected by filtration, washed with cold H 2 O, and dried in a desiccator to give, following recrystallization from ethanol, the pure diacetate product as a white solid (3.7 g, 95%).

Pomegranate peel extraction
The pomegranate was purchased from local supermarket in Mosul. The pomegranate peel was isolated from seeds manually then washed with water and dried under dark. A weight of 50 g of dried peel was smashed using a mortar, then extracted by cold percolation with 350 ml of methanol for 12 h to offer, after reducing the solvent under vacuum, the desired methanol extract of 19 g. Water (150 ml) was then added and extraction was performed with 50 ml chloroform three times to offer the CHCl 3 extract of 0.303 g. The residue of water from the first extraction above was extracted three times by adding ethyl acetate (50 ml) to yield an ethyl acetate extract of 0.73 g. 2.10 Measurement of lipase activity against the extracts or 1,4-diacetoxybenzene A volume of 0.2 mL of partially purified lipase was incubated with 0.1 mL of 1,4diacetoxybenzene (1-10 Mm) and pomegranate extracts (0.5-4 mg/ml) for 30 minutes at 37 °C separately. After that, the activity of lipase enzyme was estimated in the presence of the substrate at a concentration of 1.5 mM.

Inhibition type test
The enzymatic reaction was carried out in order to verify the type of inhibition of the partially purified lipase by incubating 0.1 ml of 1,4-diacetoxybenzene (6 mM) with 0.2 mL of the enzyme for 30 minutes [27]. The enzyme activity was estimated using different concentrations of the substrate that ranged 0.25-3 mM.

HPLC analysis
The sample was analyzed by high performance liquid chromatography, model (SYKAM) Germany. Pump model: S 2100 Quaternary Gradient Pump, Auto sampler model: S 5200, Detector: UV (S 2340), and Column Oven model: S 4115. ODS C-18 column (25 × 4.6 mm). An aliquot (10 μl) was injected and eluted at the temperature of 40°C according to Muchuweti et al. (2007) [28], using a solvent of Methanol: D.W: formic acid (70:25:5 V/V) as the mobile phase. At the wavelength of 280 nm, the analysis was accomplished at a flow rate of 1 mL/min.

Purification of lipase from pecan karnel
The results shown in Table 1 indicate that the specific activity of lipase after the sedimentation process was 1,047 units/mg protein, meaning that it was multiplied by 7.2 times. The amount of recovery of the total activity of the enzyme was 79.2% compared to the crude extract. After the precipitation process, the dialysis technique was applied as a second step in purifying the enzyme by removing ammonium sulfate used in the previous step, as well as peptides, amino acids, ions, and some small molecular weight compounds. The results shown indicate that lipase specific activity after this process is 1.66 units/mg protein, that is, it was multiplied by 10.84 times compared to that of the crude extract. After passing the protein solution, which was produced from the dialysis, through the column containing cation exchanger CM-cellulose, we obtained tow lipase isoenzyme (I, II) ( Figure 1) with specific activity values of 2.66 and 1.7 U/mg protein, respectively (Table 1). Sunflower seeds contain a high percentage of oils, which suggests the possibility of the presence of the enzyme lipase significantly. The lipase was purified from sunflower seeds by Al-Haidari and his group [29], where two peaks were shown using gel filtration chromatography. They had 4.644 and 6.482 units/mg protein. The lipase was identified from pistachio khunjuke karnel, and the purification fold was 8.216 times compared to that of the crude enzyme [30]. Priyanka et al. [31] isolated the lipase enzyme from Pseudomonas reinekei bacteria, and it was observed that one peak appeared, with a specific activity of 4.23 units/mg protein, while the activity recovery was 13.72%.

Figure 1-Purification of lipase by CM-Cellulose chromatography
The results obtained indicate that the highest activity of both purified isoenzymes was achieved at pH = 8 and 6 by using the buffer solution KH 2 PO 4 -Na 2 HPO 4 , as shown in Figures  2 and 3, respectively.    These results are close to those which found that the optimum pH of the enzyme extracted from the palm tree mesocarb is 7 [32]. It was also noted that lipase purified from pistachio khinjuke karnel had a maximum activity at pH 7.5 [30]. While the maximum activity of lipase from Aspergillus fumigatus was at pH 9 [33]. Regarding the effect of pH, as it is known, each enzyme has an optimum pH at which it shows the highest activity. At higher or lower values than the optimum pH, the secondary and tertiary structures of the enzyme molecule as a whole or of the active site of this enzyme change, thus affecting its activity. Generally, enzymes are affected by increasing or decreasing the temperature because the enzymes, due to the nature of proteins, are sensitive to thermal changes [34]. In this study, lipase activity was estimated at different temperatures, ranging 20-50 °C, and incubation time of 15 minutes. An initial increase in temperature was obtained, which led to an increase in the enzyme activity for each of the two peaks of the pecan karnel, reaching a maximum of 40 and 50 mº, as shown in Figures 4 and 5, respectively.  The results for peak I are consistent with what was found in two previous studies of purified lipase from pistachios khinjuke karnel and Aspergillus fumigatus, respectively, where the optimum temperature was at 40°C [30,33]. Okunwaye and his group [32] found that the optimum temperature of the enzyme molecule purified from a fine palm tree was 40 °C. Also, lipases were purified from yeast, where the optimal activity was achieved at 35 °C [35].
Prajapati et al. [36] reported that the extraction of lipase from the bacteria Cellulomonas  flavigena UNP3 yielded an optimally activity at 30 °C. However, the optimal activity of lipase extracted from Acinetobacter sp. was found to be reached at 50°C [37].

Molecular weight
The results indicated that the peaks of the activity of lipase extracted from the pecan karnel, which were generated by ion exchange chromatography, appeared in the form of a single clear band for each isoenzyme when applying SDS-PAGE electrophoresis technique ( Figure  1). The molecular weight of the partially purified lipase was approximately calculated to be 42 kDa for both bands ( Figure 6). This molecular weight is close to that of lipase purified from the pistachio khinjuke karnel, as its molecular weight was approximately 43.5 kDa [30]. However, it is higher than that of lipase purified from palm, which had a molecular weight of 35 kDa [32]. While it was lower than that of the enzyme purified from Pseudomonas reinekei using ion exchange chromatography, which was found to be 50 kDa [31].

Preparation of 1,4-diacetoxybenzene
A literature search disclosed several methods described by Prichard, in which 1,4diacetoxybenzene could be prepared by adding acetic anhydride to hydroquinone in the presence of sulfuric acid, to give, following recrystallization from ethanol, the desired product in 95% (Scheme 1) [25]. of acetyl group at δ= 2.29 and lacked the characteristic OH signal at δ= 8.6 [38] for hydroquinone (Figures 7 and 8).

Lipase inhibition
The inhibitory effect of 1,4-diacetoxybenzene at different concentrations (1-10 mM) on the activity of the purified lipase was investigated. Table 2 shows that these concentrations led to a decrease in the activity of the enzyme compared to its activity in the absence of the inhibitor. The highest percentage of enzyme inhibition was 38.3% at 6 mM concentration. Accordingly, this concentration was adopted in the study of the type of inhibition.

Inhibition mode
The inhibition of the purified lipase activity was studied by the presence of the inhibitor 1,4diacetoxybenzene at a concentration of 6 mM. The inhibition mode was verified by drawing Lenweaver-Burk plot ( Figure 9)  The compound 1,4-diacetoxybenzene prepared in the laboratory is a diester compound, as it contains two ester groups in the Para site. This basis can explain that the type of inhibition of this compound was competitive inhibition. In a previous study, the inhibition type was found to be non-competitive for the lipase extracted from the pistachio khinjuke karnel, by using the two compounds of Quercetin rutinoside and Melatonin, where the values of Ki were 1.025 and 1.006 mM, respectively [30]. It was also revealed that the extract of Levisticum officinale showed mixed inhibition of pancreatic lipase enzyme, while the inhibition was noncompetitive when using extracts of Quercus infectoria, Eucalyptus galbie, and Rosa damascene [39]. Also, the inhibition of lipase enzyme isolated from the fungus Candida

Non inhibition
With inhibitor [6]mM rugosa and wheat seeds were followed up by polysaccharide chitosan, and it was found that the inhibition constant values were 1.4 and 0.9 mM, respectively [40]. Effects of pomegranate peel extracts on the activity of lipase enzyme Table 3 shows the effects of chloroform extract on enzyme activity, as the highest percentage of the extract effect was at a concentration of 0.2 mg/ml and the lowest at a concentration of 0.4 mg/ml.  Table 4 also shows the effects of ethyl acetate extract on enzyme activity, where the highest percentage of the extract effect was at a concentration of 0.6 mg/ml and the lowest percentage was at 0.8 mg/ml The results of HPLC analysis (Figures 10 and 11) indicate the appearance of several bands belonging to the chromatograms of standard phenolic compounds (gallic acid, chlorogenic acid, tannic acid, para-coumaric acid, and quercetin) with concentrations and retention times specific to each compound, as shown in Tables 5 and 6.   It is evident from the two tables that the concentration of para-coumaric acid was the highest in the two extracts, reaching 89.84 and 12.38 mg/g, respectively, while quercetin was the lowest, reaching 11.39 and 0.177 mg/g, respectively. Pomegranate peel is a rich source of tannins, flavonoids, and other phenolic compounds [41][42]. HPLC analysis was used in a previous study to identify phenolic compounds in pomegranate peel extracts using ethanol and isopropanol. Polyphenolic compounds, such as p-coumaric acid, caffeic acid, ellagic acid, cinnamic acid, quinic acid, and iso-ferulic acid, were discovered [13]. The extraction of various phenolic compounds from pomegranate peels was demonstrated [43]. Mansour et al. [44] exhibited the occurrence of caffeic, ellagic, vanillic, gallic, and p-coumaric acids as well as quercetin in pomegranate peel extract. HPLC was used to find chlorogenic acid, ferulic, coumaric, and cinnamic acids in pomegranate peel methanolic extract [18].

Conclusions
In the current study, two peaks of lipase were purified and characterized from pecan karnel by using paranitro phenyl acetate as substrate. The molecular mass of both isoenzymes was found to be 42 kDa by using electrophoresis. The enzyme was inhibited competitively by using 1,4-diacetoxybenzene compound, but activated with pomegranate peel extracts. Also, some phenolic compounds were identified in pomegranate peel extracts by HPLC technique. Several peaks appeared that belong to the chromatograms of gallic acid, chlorogenic acid, tannic acid, para-coumaric acid and quercetin, with concentrations and retention times specific to each compound.