INTRODUCTION:

Enzymes are catalysts which are responsible for much metabolic activity that maintain life. Enzymes are accelerating the rate and specificity of metabolic reactions starting the digestion of food to the DNA synthesis¹. The major goal of the food industry is to decrease the deterioration reactions. During the processing of tomatoes polyphenoloxidase enzymes can cause several changes such as nutritional properties, color, aroma composition and reduction of provitamin A². Many fruits and vegetables turn out to be discoloured during storage and processing due to presence of enzyme polyphenol oxidase³. Polyphenol oxidases are present among bacteria, fungi, archaea, plants, insects and animals and its contain a type-III copper.

The quinones formed due to non-enzymatic reactions complex polymers by PPOs are highly reactive known as melanins. The presence of these dark brownish substances reduces the quality and price of many fresh fruits⁴. They freely presented of ripening plants in cytoplasm. Polyphenol oxidases show resistance effects against microbial and viral infection in plants and also present in animals to increases disease resistance in insects as well as crustaceans. Polyphenol oxidases present in tomato, work as the defense mechanism toward diseases, whereas PPO enhances its resistance towards pathogenic microorganisms⁵.

The present study was focused on degradation of industrially used azodyes through Polyphenol oxidase enzyme extracted from tomato peel and pulp. There is further investigation will be required to analysis of molecular weight of enzyme. PPO has a potential to develop as a degrading agent for the reduction of organic pollutants.

MATERIAL AND METHODOLOGY:

Sample Collection:

The tomato samples were collected from the local market in Raipur, Chhattisgarh. They were processed carefully to remove foreign contaminants and dirt. The peel and pulp of the tomato were then separated by using a sterile scalpel.

Extraction of Crude Polyphenol oxidase:

The crude Poly phenol oxidase extracted from peel and pulp of tomato using 0.05M potassium Phosphate Buffer (pH 6.5). The peel and pulp were separately crushed with 0.05M Potassium Phosphate Buffer containing 10mM ascorbic acid and 0.5% Polyvinyl pyrrolidone (PVPP). Then filtered and centrifuged at 3000g for 20 minutes at 4°C⁶.

Partial Purification:

The Polyphenol oxidase enzyme was then extracted from the crude peel and pulp extract by salt precipitation method using Ammonium sulphate salts. The salt precipitated sample was precipitated upto 80% saturation. The precipitated enzymes are then isolated by centrifugation at 3000g for 10Min. The salt precipitated sample was dialysed using Dialysis bag-110⁷.

Ion Exchange Chromatography:

The dialysis samples were purified by ION Exchange chromatography using Carboxymethyl cellulose as stationary phase. Gradient type Elution buffers were used. A total six elutes were prepared containing a single buffer system (50mM Potassium phosphate buffer, pH-6.5) and the six elution buffers contained increasing concentration of NaCl (25mM, 50mM, 75mM, 100mM, 125mM, 150mM)⁸.

Quantitative estimation of Protein:

For determination of protein concentration in samples, a standard calibration curve of Bovine serum albumin was established. The standard calibration curve has absorbance at 660nm. By plotting the absorbance of the sample in the slope equation (y= mx+c) as deduced by BSA calibration curve, we got the concentration of protein in sample⁹.

Determination of Enzyme activity and Specific activity (Catechol oxidase):

The Enzyme activity assay was carried out for crude enzyme sample, salt precipitation sample, dialysis samples and elutes from ion exchange chromatography. The absorbance for the test and blank solution was observed until constant. To test solution, 0.10ml enzyme sample was mixed to the test solution. The change in Absorbance (A_265nm) was recorded for approximately 5 minutes for both Test and Blank and recorded by using maximum linear rate^10,11.

The enzyme activity (Units/mL) was calculated as

Enzymatic activity of enzyme (Units per ml) =

ΔA265nm/minute TEST - ΔA265nm/minute BLANK × df

----------------------------------------------------------------------------

(0.001) (0.1)

Where

Df = Dilution factor for the enzyme solution.

0.001 = The change in A_265nm/minute per unit of Catechol Oxidase

0.1 = Volume of enzyme used (in mL)

The specific activity of the enzyme (Units/mL)

Specific activity of enzyme (Units per mg) =

ΔA265nm/minute TEST - ΔA265nm/minute BLANK × df

----------------------------------------------------------------------------

(0.001) (mg enzyme / RM)

Where,

0.001 = The change in A_265nm/minute per unit of Catechol Oxidase

mg enzyme= Quantity of enzyme present in the reaction mixture.

RM = Reaction Mix (final volume = 3 ml)

Decolourization assay:

The purified enzymes from peel and pulp of tomato by ion exchange chromatography were analyzed for degradation of different azodye. Five dyes Congo Red, MethylYellow, Auromine, Rhodamine, Methyl Violet were used. Each dye (150µg mL⁻¹ ) was incubated with Tomato PPO in 50 mM phosphate buffer, pH 6.5 at 30◦C. The disappearance of the color was monitored at λmax of the respective dye solution. The percent decolorization was calculated by follow calculation¹².

Percentage of degradation =

Abs (Control) – Abs (Test)

---------------------------------------------------------------------× 100

Abs (Control)

Where Abs (control) = Absorbance of control, Abs (Test) = Absorbance of Test

RESULT AND DISCUSSION:

Extraction and Purification of PPO:

The Polyphenol oxidase enzyme was extracted from the crude peel and pulp of tomato and precipitated by using 80% concentration of Ammonium salts. The dialysis was carried out for 5-6 days for diffusion process change every 12hours of dialysate buffer. The principle behind of changing of dialysate buffer is to enable the process of diffusion of protein so as the purity level. The dialysis sample of peel and pulp were purified by cationic exchanger. Elution of a protein of interested was accomplished by increasing concentration of NaCl in the column. NaCl is usually used due to chloride anions attach to the resin and transfer the protein. A suitable buffer system brings the pH close.

Quantitative Estimation of Protein:

The quantitative estimation of protein was estimated from the BSA standard curve by putting the absorbance of samples in the slope equation y=0.006x + 0.077 to find out the concentration of unknown. The protein concentration of sample was first analyzed with 0.1ml of sample and 0.9mL of distilled water. So in order to get the actual protein concentration, dilution factor is to be multiplied with the concentration that we got for 0.1mL of sample as dilution factor (df) = Total volume/Aliquot volume = 1000µL/100µL= 10. Elute 3 for peel has observed highest concentration of protein 0.18mg/ml and Elute 4 for pulp was observed 0.21mg/ml.(mg/mL equivalent of BSA) (Graph 1)

Graph 1: Bovine Serum Albumin Standard Curve

Enzyme activity and Specific activity:

The Enzyme activity and specific activity assay was performed for crude enzyme, salt precipitation, dialysis and purified polyphenol oxidase enzyme elutes 3 (Peel) and Elute 4 (Pulp). The linear curve for samples was prepared.

Graph 2: Protein Concentration of Peel and Pulp Enzyme

Graph 3: Maximum linear rate curve for blank.

Graph 4: Maximum linear rate curve for crude enzyme from peel of tomato.

Graph 5: Maximum linear rate curve for crude enzyme from tomato pulp.

Graph 6: Maximum linear rate curve for Salt precipitation enzyme from tomato peel.

Graph 7: Maximum linear rate curve for Salt precipitation enzyme from tomato pulp.

Graph 8: Maximum linear rate curve for Dialysis enzyme from tomato peel.

Graph 9: Maximum linear rate curve for Dialysis enzyme from tomato pulp.

Graph 10: Maximum linear rate curve for Ion exchange Elution 3 enzyme from tomato peel.

Graph 11: Maximum linear rate curve for Ion exchange Elution 4 enzyme from tomato pulp.

The above graphs, showed decrease in absorbance so the slope m is negative. The slope represents the change in absorbance per minute quantitatively. The mean slope for blank and enzyme samples for change in absorbance/per minute was presented in table 1.

Table 1: Mean slope for different samples of enzymes(crude enzyme, partial purified and purified enzymes).

Sample	Mean Slope m = (∆A_265nm/minute)
BLANK	0.0028
Crude enzyme (Peel)	0.0072
Crude enzyme from Pulp	0.0041
Salt ppt. Tomato peel	0.0093
Salt ppt. Tomato pulp	0.0073
Dialysis enzyme of Tomato peel	0.0097
Dialysis enzyme of Tomato pulp	0.0107
Elution 3 Tomato peel	0.0148
Elution 4 Tomato pulp	0.0070

The specific activity of crude enzymes peel and pulp were 16.296 U/mg and 5.0 U/mg recorded respectively. In the partial purified, dialysed enzymes were 34.5 U/mg and 41.579 U/mg observed respectively for peel and pulp. However, purified elute 3 of peel by ion exchange chromatography found out higher specific activity 240.0 U/mg from pulp.

Table 2: Specific activity of different enzyme samples from peel and pulp of tomato.

Samples	Protein Concentration (mg/mL).	Specific Activity (Units/mg)	Enzyme Activity (Units/ml)
Crude (Peel)	0.82	16.296	44.0
Crude (Pulp)	0.79	5.000	13.0
Salt ppt (Peel)	0.63	30.952	65.0
Salt ppt (pulp)	0.61	22.500	45.0
Dialysis (Peel)	0.61	34.500	69.0
Dialysis (Pulp)	0.59	41.579	79.0
Ion Exchange (Elution 3) Peel	0.14	240.000	120.0
Ion Exchange (Elution 4) Pulp	0.19	70.000	42.0

The enzymatic activity of crude peel and pulp enzymes was 65.0U/ml and 13.0U/ml presented by catechol oxidase respectively. Whereas partial purified dialysed enzymes were 69.0U/ml and 79.0U/ml respectively for peel and pulp. By ion exchange chromatography purified etute 3 of peel found out much greater than pulp with 120.0U/ml.

Graph 12: Specific activity and enzymatic activity of different enzyme samples from peel and pulp of tomato.

Decolourization assay:

The enzyme extracts (purified enzymes from peel and pulp of tomato) by ion exchange chromatography were analyzed for degradation against Congo Red, Methyl Yellow, Auromine, Rhodamine, Methyl Violet azodye. The Ion exchange Elution 3 for peel was taken and where as the elution 4 were taken from purified enzyme from pulp for degradation. The five dyes were assayed at different wavelength at their corresponding Absorbance maxima (ƛmax) as described in Table: .

Table 3: Absorbance maxima (ƛmax) for different azodyes

Dye	ƛmax in nm
Auromine	438 nm
Congo Red	498nm
Methyl Yellow	410 nm
Methyl Violet	590nm
Rhodamine	410nm

The purified elute 3 of peel investigated highest degradation 80.76% against auromine as well as lowest degradation against rhodamine. On other hand, pulp purified elution 4 showed 73.08% degradation against auromine.

Graph 13: Degradation of dyes though purified elute of peel and pulp.

CONCLUSION:

The tomato peel and pulp Polyphenol Oxidase has an immense capability for the decolorization of industrial dyes. It does not require any kind of redox mediators, low cost process. Polyphenol Oxidase enzyme consists oxidative cleavage of the dyes as well as not leaving any kind of mutagenic and toxic substance. The polyphenol oxidase may be further used as degradative agent in the textile industries.

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Received on 24.07.2023 Modified on 15.03.2024

Research J. Pharm. and Tech 2024; 17(9):4242-4246.

DOI: 10.52711/0974-360X.2024.00655