Characterization of Catalase Enzyme from Leaf Tissue of Aronia (Aronia melanocarpa) Plant

DOI: https://doi.org/10.21203/rs.3.rs-2309242/v1

Abstract

Aronia is among the most antioxidant containing plants which is found commonly around the worls. Aronia cultivation started in Turkey for the first time in 2012 at the Atatürk Central Research Institute of Garden Cultures, and a plantation was constructed in the experimental area. Since antioxidants help to preserve food by blocking oxidation processes and contributing to the health promotion provided by numerous dietary supplements, nutraceutical and functional food additives, antioxidant capacity of these plants should be well characterized. To assess and evaluate the antioxidant content of foods and plant products, many approaches are utilized. In this study, catalase enzyme was partially purified from aronia plant leaf tissue and characterization was carried out. Purification process consisted of homogenate preparation, ammonium sulfate precipitation and dialysis. The optimal ionic strength, pH, substrate concentration and enzyme quantity were examined. These values were found to be 300 mM TRIS, pH:8, 12 mM H2O2 and 75 µl, respectively, for the catalase enzyme of the Aronia plant leaf tissue. This study is the first in the literature dealing with the characterization of antioxidant enzyme from Aronia plant.

1. Introduction

A native plant of North America, Aronia melanocarpa (aronia) is commonly known as black chokeberry and is now cultivated worldwide. The aronia plant, which belongs to the Rosaceae family, is very rich in anthocyanins and other phenolic compounds and has a dark purple color. With its high total polyphenol and anthocyanin content and DPPH radical scavenging activity, Aronia is known to have powerful antioxidant features compared to many other fruits [1, 2].

Aronia has a wide range of phenolic chemicals, including phenolic acids, flavonols, anthocyanins, and flavan-3-ols[3]. So far, it has been shown that aronia berry decreases cystolic blood pressure and cholesterol levels [4], which reduces the risk of chronic illnesses [5] and provides strong antioxidant protection [6].

O2 is generally not reactive to most cellular components, but ROS (reactive oxygen species) causes oxidation of lipids, proteins, RNA, DNA and many small molecules in the cell. ROS have a high reactivity to these biological components due to their changed chemistry as compared to O2, which permits them to donate an electron or transfer an excited energy state to an acceptor molecule [7]. Hydrogen peroxide (H2O2), superoxide (O2), singlet oxygen (1O2), the hydroxyl radical (HO.) and different types of organic and inorganic peroxides are the principal forms of ROS in cells, which vary widely in their characteristics and chemical reactivity [711]. Since ROS is very reactive and is produced independently in nearly all cell compartments, its levels must be controlled to prevent undesired cellular oxidation.

A group of polyphenolic compounds commonly found in fruits, vegetables and other food products and produced as secondary metabolites in plants are called flavonoids. In addition to other bioactivities (e.g., anti-inflammation, anti-aging), flavanoids have beneficial biochemical effects on certain diseases (eg cardiovascular disease, atherosclerosis) [1214]. Their principal biological function is antioxidant protection. Flavonoid antioxidant activity can protect against free radical damage by scavenging reactive oxygen species, activating antioxidant enzymes, inhibiting oxidases (e.g., xanthine oxidase [XO], cyclooxygenase [COX], lipoxygenase and phosphoinositide 3-kinase [PI3K]), and reducing α-tocopheryl radicals. To decrease oxidative stress, flavonoid antioxidant activity can raise uric acid levels, metal-chelating activity, and low-molecular-weight antioxidant activity [13].

Antioxidants have been promoted as helpful agents in improving plant stand and minimizing the impacts of biotic and abiotic stressors.

Plants have many enzymatic and non-enzymatic defensive strategies against oxidative stressors caused by ROS. The antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) have an important place in the enzymatic defense systems of plants to remove ROS [15]. SOD is the primary O2. scavenger and results in the formation of H2O2 and O2 by enzymatic reaction. The H2O2 generated is subsequently removed by CAT [16].

Catalase (H2O2: H2O2 oxidoreductase E.C.1.11.1.6) is a kind of antioxidant enzyme found in all aerobic organisms. It is known that H2O2 is converted into water and oxygen in the cell in the presence of catalase with the realization of environmental stress. Catalase is found in all major locations of H2O2 generation in higher plants' cellular environments (such as peroxisomes, mitochondria and cytosol) [17].

Due to the importance of catalase in plant defense system, we aimed in this study to purify the enzyme from Aronia melanocarpa leaves for the first time and to determine optimum buffer, optimum ionic strength, optimum pH and optimum substrate amount in order to find new potential natural antioxidant sources.

2. Methods

2.1. Chemicals

Sigma-Aldrich supplied the chemicals utilized in the purifying process Aldrich (St. Louis, Mo, USA). Merck supplied the other compounds used (Darmstadt, Germany).

2.2. Preparation of the homogenate and in vitro enzyme assay

Leaf tissue was obtained from the aronia plant. The leaves were crushed into small pieces and then thoroughly crushed and homogenized with liquid nitrogen (approximately − 196°C), then 300 mM TRIS buffer was added and centrifuged at 4°C, 15,000 xg. After centrifugation step, the supernatant was filtered and enzyme activity was measured. The activity was measured at 240 nm using a Shimadzu UV-1800 spectrophotometer.

Hydrogen peroxide (H2O2) was used as the substrate for catalase. 100 µl of H2O2 in 0.3 M Tris buffer (pH 8.0) was transferred to the cuvette. The enzymatic reaction was initiated by adding 100 µl of supernatant containing catalase enzyme to the cuvette, and the final volume was reduced to 1 ml with distilled water; the absorbance value at the commencement of the reaction was then measured in a spectrophotometer at 240 nm.

2.3. Ammonium sulphate precipitation and dialysis

Ammonium sulfate precipitation was performed for the prepared homogenate. Accordingly, the homogenate was adjusted at different intervals with solid ammonium sulfate at a salt concentration of 0-100%. The precipitate was dissolved with a minimal amount of tris buffer 0.3 M, Tris). The maximum enzyme activity was found at concentrations ranging from 20–40%.

To remove salts from protein solutions, dialysis was performed. A dialysis bag with a semi-permeable membrane, typically composed of cellulose acetate and having a porous structure, is utilized for this procedure. The prepared solution was placed in this bag and slowly mixed by passing it through the suitable buffer. Small molecules go across the membrane until the osmotic pressure was adjusted. The buffer outside the membrane was altered multiple times during this procedure.

2.4. Characterization of the enzyme with kinetic parameters

To characterize the enzyme, different pH, substrate, and ionic strength parameters were examined. The enzyme's characterization parameters were calculated as optimum ionic strength: 300 mM Tris, pH: 8 and substrate concentration: 12 mM.

3. Results

Catalase enzyme was partially purified and characterized from aronia plant leaf tissue in this work. The high antioxidant content of the aronia plant and its favorable effects on human health highlight the significance of our work. Many plant leaves have been used to make herbal tea and contain high levels of phytochemicals derived from polyphenols, flavonoids, and chlorophyl. Phytochemicals are secondary metabolites present in plants that have been extensively researched for anticancer, anti-inflammatory, and antibacterial properties [18, 19]. Quercetin, rutin, and chlorogenic acid are the most abundant polyphenols in aronia leaves [20]. Polyphenols are the most abundant antioxidant chemicals in plants which can reduce inflammation, cancer, and aging by quenching reactive oxygen species [21].

The characterisation research is critical for determining and selecting the ideal values for the importance and antioxidant characteristics of the aronia plant-derived catalase enzyme. As previously stated, in addition to the relevance of the aronia plant's leaf content, the characterisation of the catalase is critical for both the plant and the enzyme.

Because of the strong antioxidant content of aronia, the purification and characterisation of the catalase enzyme demonstrates the study's uniqueness.

Catalase enzyme was partially purified from aronia plant and characterized in this work for the first time. After homogenization, the enzyme's precipitate saturation with solid (NH4)2SO4 was determined to be 20–40%. This finding demonstrates that the purification process is consistent with previous investigations and will serve as a model for future research. Both potassium phosphate and tris buffer measurements were made for optimum ionic strength optimization. The ideal ionic strength was evaluated between 10 mM and 600 mM Tris buffer as a result of the optimization experiments, and the optimum ionic strength was identified in 300 mM Tris (Table 1). pH was adjusted between 5 and 8,5 to find the optimal pH, which was determined to be 8 (Table 2). In addition, the optimum substrate concentration was measured between 3 and 15 mM and the optimum substrate concentration was found to be 12 mM H2O2 (Table 3).

4. Discussion

Many studies on the inhibition of CAT and other antioxidant enzymes have been done in the literature, and the results showed similar results with our study.

In a study by Dinçler and Aydemir, catalase enzyme was purified from the chard plant. As a result of the study, a wide optimum pH range was found to be 6.0–8.0. At the same time, the precipitation range of ammonium sulfate was found to be 45% [22].

In another study, catalase enzyme was purified from sprouted black gram (Vigna mungo) seeds and optimum pH and temperature were found to be 7.0 and 40°C, respectively [23].

In the purification and characterization study of the catalase enzyme carried out in Turkey Van apple, the optimum pH value was found to be 5 and the optimum temperature was 50°C [24].

In the study of partial purification of catalase enzyme from red cabbage, the optimum pH was found to be 7 at an optimum temperature of 30°C [25].

Agaricus bisporus is a well-known and extensively consumed mushroom. The best pH value for the purification and characterisation of the catalase enzyme from this fungus was determined to be 7.5, while the optimum ammonium sulfate precipitation range was 45–90% [26].

Catalase enzyme is found not only in plants but also in animals and the purification process takes place. Purification and characterization studies from different animals and different tissues have also found similar results with our study.

In the study on the purification and partial characterization of catalase from chicken erythrocytes, the optimum pH was 7 and the optimum temperature was 25°C [27].

The optimal pH and temperature in the Purification and Properties of Liver Catalase in Water Buffalo (Bubalus bubalis) investigation were determined to be 7.5 and 30 C, respectively [28].

In a research comparing purification and characterisation of liver catalase with normal dog liver catalase in the acatalasemic beagle dog, the activities of wild type and acatalasemic dog liver catalases revealed distinct pH profiles in the pH range of 3 to 11. Catalase activity purified from wild type dog liver did not alter significantly across a large pH range, although it did demonstrate activity even at pH 11. Catalase activity isolated from acatalasemic dog liver, on the other hand, was only stable in a restricted pH range of 6–9 [29].

Catalase enzyme has been isolated from many tissues of both plants and mammals, as observed in our work and other investigations, and characterisation tests have been performed. We think that the results obtained from our study will contribute to catalase enzyme purification and characterization studies to be carried out in the future.

5. Conclusions

As a result, catalase enzyme was characterized from leaf tissue of aronia plant. This study is the first to reveal the partial purification and characterization of the catalase enzyme of aronia, which is an economically important plant and has high antioxidant value in both the leaf and fruit part. Our findings will help to promote the emergence of novel aronia plant characteristics as well as the consumption of the plant.

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Tables

Table 1. Activity measurements of aronia fruit leaf tissue catalase enzyme optimal ionic strength TRIS buffer

[mM]              10      20         50     100       150    200    300       400    500    600

%Activity     22.8   74.8       64.5  58.2       84     70.8   100       53.7      74.8   63.4

Table 2. Activity measurements of aronia fruit leaf tissue catalase enzyme optimal pH value Tris (300 mM) buffer

pH                  5.0        6.0        6.5        7.0        7.5        8.0       8.5     

%Activity      95.2     62.8         56.2      68.8      82.0      100         90.4

Table 3. Optimum substrate concentration 300 mM TRIS (pH=8) buffer activity measurements for aronia fruit leaf tissue catalase enzyme

H2O2 (mM)          3            6           9           12            15

%Activity           25           50        75          100           125