"Optimizing Guaiacol Oxidation: Activators of Watermelon Peel Peroxidase"

Abstract

The invention relates to a method for enhancing the activity of peroxidase enzymes extracted from watermelon peel, utilizing metallic chlorides as activators in the oxidation of guaiacol. The study investigates the effects of metallic chloride salts, including CuCl₂, FeCl₃, KCl, and MgCl₂, at concentrations ranging from 0.5 mM to 2 mM. The reaction mixtures consist of crude peroxidase extract, sodium acetate buffer (pH 5.4), guaiacol, hydrogen peroxide, and the respective metallic chloride salt. The enzyme activity is measured spectrophotometrically at 470 nm by monitoring guaiacol oxidation. Results demonstrate that all tested metallic chlorides enhance peroxidase activity, with each exhibiting an optimal concentration for maximum activation. This invention provides a sustainable and cost-effective approach for utilizing agricultural waste, such as watermelon peel, to develop efficient biocatalysts with potential applications in industrial processes, environmental remediation, and biotechnological advancements.

Specification

Description:


Field of the Invention:
[001] The field of the invention pertains to enzymology and agricultural biotechnology, focusing on the activation of peroxidase enzymes extracted from agricultural byproducts, specifically watermelon peel, using metallic chlorides. It delves into enzyme catalysis and the enhancement of peroxidase activity, particularly in the oxidation of guaiacol. This research aligns with sustainable agricultural practices and waste valorization by utilizing watermelon peel as an alternative source of enzymes. Additionally, it contributes to the development of cost-effective biocatalysts for industrial and environmental applications, highlighting the role of metallic chlorides (e.g., CuCl₂, FeCl₃, KCl, and MgCl₂) in optimizing enzyme performance. This work advances enzyme technology by providing insights into effective activation methods and promoting sustainability in biochemical processes.
Background of the invention and related prior art:
[002] Peroxidase enzymes play a crucial role in various biochemical processes, including oxidation reactions, and have wide applications in industries such as biotechnology, pharmaceuticals, and environmental remediation. Traditionally, peroxidases are sourced from expensive or limited biological materials, prompting the need for alternative, cost-effective sources. Agricultural byproducts, such as watermelon peel, offer an abundant and sustainable resource for enzyme extraction, aligning with global efforts to reduce waste and promote circular economy practices. However, optimizing the activity of such enzymes is essential to maximize their utility. Previous studies have shown that metallic salts can act as activators for enzymes, enhancing their catalytic performance. This invention focuses on the activation of watermelon peel peroxidase using metallic chlorides (Cu, Fe, K, and Mg), aiming to identify optimal conditions for enzyme activity, thus contributing to the development of sustainable and economically viable biocatalysts.
[003] A patent document US9205115B2 discloses methods of inducing systemic acquired resistance to infection in a plant. The methods comprise applying a composition comprising a Bacillus control agent to said plant wherein said plant is capable of producing defense proteins. Also provided are, methods for controlling one or more plant diseases, methods for preventing plant virus transmission, methods for preventing and/or treating soil-borne plant pathogens using the Bacillus control agent of the present invention, and methods of generating bacterial spores. In addition, synergistic biocontrol combinations and methods of using the same are provided.
[004] Another patent document TWI801925B disclosed compositions used and methods for regulation of immunity homeostasis including a combination of an Aloeextract enriched for one or more polysaccharides; a Poriaextract enriched for one or more polysaccharides; and a Rosemaryextract enriched for one or more polyphenolic compounds. Compositions for maintenance of immunity homeostasis by regulating HMGB1, comprising a combination of one or more polysaccharides and one or more polyphenolic compounds are disclosed. Methods for treating, managing, promoting regulation of immunity homeostasis in a mammal are disclosed that include administering an effective amount of a composition from 0.01 mg/kg to 500 mg/kg body weight of the mammal.
[005] A document CN116941679A discloses a perilla seed oil with high antioxidant activity and a preparation method thereof, wherein the content and the proportion of specific antioxidant active ingredients rosmarinic acid and tocopherol in the perilla seed oil are taken as research objects, the proportion of the specific antioxidant active ingredients rosmarinic acid and tocopherol with an antioxidant synergistic effect is obtained, and the obtained perilla seed oil product has excellent antioxidant effect and also has good oil nutrition value and sensory quality, so that a new research idea is provided for optimizing oil processing conditions and improving the quality of oil products.
[006] Another document CN101575381A discloses a no-waste production method for peeled sweet potato starch. The provided method comprises the following steps: step 1. the sweep potato is peeled and/or the speck is removed; step 2. the sweep potato obtained in the step 1 is squeezed to obtain coarse size and coarse potato residue; and step 3. the coarse size obtained in the step 2 is treated to obtain starch in the coarse size and a first by-product, i.e., sweet potato size; the coarse potato residue obtained in the step 2 is treated to obtain starch in the coarse potato residue and a second by-product, i.e., potato powdered residue; and the coarse size and the washing water obtained during the coarse potato residue treatment are treated to obtain a third by-product containing polysaccharide and protein and water, and the water can be further taken as the washing water for the sweet potato, the starch and the coarse potato residue. The method can utilize all the nutritious substances in the sweet potato and is free from the influence of reduction of the starch content during the storage process of the sweet potato, and the application of the sweet potato size and the concentrated solution of the washing water provides a possibility of prolonging the processing cycle of the sweet potato starch.
[007] None of these above patents, however alone or in combination, disclose the present invention. The invention consists of certain novel features and a combination of parts hereinafter fully described, illustrated in the accompanying drawings, and particularly pointed out in the appended claims, it being understood that various changes in the details may be made without departing from the spirit, or sacrificing any of the advantages of the present invention.
Summary of the invention:
[008] The invention investigates the activation of peroxidase enzymes extracted from watermelon peel using metallic chlorides, specifically CuCl₂, FeCl₃, KCl, and MgCl₂, in the oxidation of guaiacol. Employing spectrophotometric analysis at 470 nm, the study evaluates the effect of varying concentrations of these metallic chloride salts (0.5 mM to 2 mM) on enzyme activity. Reaction mixtures consisting of sodium acetate buffer, guaiacol, crude peroxidase extract, metallic chloride salt, and hydrogen peroxide were prepared, and the rate of guaiacol oxidation was monitored. Results demonstrated that all tested metallic chlorides effectively enhanced peroxidase activity, with each salt exhibiting an optimal concentration for maximum activation. This research highlights the potential of watermelon peel as a sustainable source of peroxidase and underscores the importance of metallic chlorides in optimizing enzyme performance for industrial and environmental applications.
Detailed description of the invention with accompanying drawings:
[009] For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing a preferred embodiment thereof, from an inspection of which, when considered in connection with the following description, the invention, its preparation, and many of its advantages should be readily understood and appreciated.
[010] The principal object of the invention is to develop optimizing guaiacol oxidation: activators of watermelon peel peroxidase. The invention focuses on optimizing the activity of peroxidase enzymes extracted from watermelon peel using metallic chlorides (CuCl₂, FeCl₃, KCl, and MgCl₂) as activators. The study evaluates the effect of these activators on the oxidation of guaiacol, a model substrate, by analyzing enzyme kinetics through spectrophotometry at 470 nm.
Materials and Methods:
1. Preparation of Reagents and Solutions:
Sodium Acetate Buffer:
Prepare 2.3 mL of 0.6 M sodium acetate buffer at pH 5.4.
Guaiacol Solution:
Use 0.2 mL of 0.02 mM guaiacol solution as the substrate for the enzymatic reaction.
Crude Peroxidase Extract:
Extract crude peroxidase from watermelon peel using standard extraction methods, such as homogenization and filtration. Use 0.1 mL for each reaction.
Metallic Chloride Salts:
Prepare solutions of CuCl₂, FeCl₃, KCl, and MgCl₂ in concentrations ranging from 0.5 mM to 2 mM. These salts are tested individually as potential activators.
Hydrogen Peroxide (H₂O₂):
Prepare 0.2 mL of 2 mM H₂O₂ solution to initiate the oxidation reaction.
2. Reaction Setup:
Control Reaction:
Prepare a control mixture without metallic chloride salts, replacing the chloride solution with an equivalent volume of distilled water.
Test Reactions:
For each metallic chloride salt, prepare a reaction mixture comprising the following components:
2.3 mL of 0.6 M sodium acetate buffer (pH 5.4)
0.2 mL of 0.02 mM guaiacol solution
0.1 mL of crude peroxidase extract
0.2 mL of the respective metallic chloride salt solution (at varying concentrations: 0.5 mM, 1 mM, 1.5 mM, and 2 mM)
0.2 mL of 2 mM H₂O₂ (added last to initiate the reaction)
Total Reaction Volume:
Each reaction mixture has a final volume of 3 mL.
3. Spectrophotometric Analysis:
Measure the oxidation of guaiacol by monitoring the change in absorbance at 470 nm using a spectrophotometer.
Record absorbance every 2 seconds for 1 minute after the addition of H₂O₂ to determine the initial velocity of the reaction.
4. Data Analysis:
Calculate the initial velocity of the enzymatic reaction by determining the slope of the linear portion of the absorbance-time curve.
Compare enzyme activity across different concentrations of metallic chlorides to identify the optimal activator and its concentration.

Results and Observations:
All tested metallic chlorides enhanced the activity of watermelon peel peroxidase.
Each chloride salt demonstrated an optimal concentration for maximum activation.
For example:
CuCl₂ showed the highest activation at 1.5 mM.
FeCl₃ achieved peak activity at 1 mM.
KCl and MgCl₂ exhibited optimal activation at 2 mM.
[011] This invention provides a sustainable and cost-effective approach to utilizing agricultural waste (watermelon peel) as a source of peroxidase enzymes. The use of metallic chlorides as activators not only enhances enzyme performance but also broadens the potential industrial and environmental applications of these enzymes. By optimizing enzyme activity, this research contributes to advancements in enzyme technology, biocatalysis, and waste valorization.
[012] Without further elaboration, the foregoing will so fully illustrate my invention, that others may, by applying current of future knowledge, readily adapt the same for use under various conditions of service.It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention.
Advantages over the prior art
[013] Optimizing guaiacol oxidation: activators of watermelon peel peroxidase proposed by the present invention has the following advantages over the prior art:
1. Sustainability:
Utilizes watermelon peel, an agricultural waste product, as a source of peroxidase, reducing waste and promoting environmental sustainability.
2. Cost-Effectiveness:
Offers an economical alternative to expensive commercial peroxidases by using a readily available and low-cost raw material.
3. Enhanced Enzyme Activity:
Demonstrates the activation of watermelon peel peroxidase using metallic chlorides, optimizing its catalytic efficiency for various applications.


4. Eco-Friendly Solution:
Reduces reliance on synthetic catalysts, offering a greener alternative for industrial and environmental processes.
5. Broad Applicability:
The optimized peroxidase can be applied in industries such as pharmaceuticals, wastewater treatment, biosensors, and bioremediation.
6. Simple Methodology:
Employs straightforward experimental protocols that are easy to replicate and scale for industrial applications.
7. Customizable Optimization:
Allows fine-tuning of metallic chloride concentrations to achieve maximum enzyme activity, making it adaptable for specific industrial needs.
8. Waste Valorization:
Adds value to agricultural byproducts, promoting the circular economy and contributing to sustainable development.
9. Industrial and Environmental Impact:
Potential to enhance biocatalytic processes in manufacturing, pollution control, and renewable energy sectors.
10. Advancement in Enzyme Technology:
Contributes to the field of enzymology by exploring alternative enzyme sources and activators, paving the way for further research and innovation.
[014] In the preceding specification, the invention has been described with reference to specific exemplary embodiments thereof. It will be evident that various modifications and changes may be made thereunto without departing from the broader spirit and scope of the invention as set forth in the claims that follow. The specification and drawings are accordingly to be regarded in an illustrative rather than restrictive sense.Therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation. The actual scope of the invention is intended to be defined in the following claims when viewed in their proper perspective based on the prior art.
, Claims:We claim:
1. Optimizing guaiacol oxidation: activators of watermelon peel peroxidase comprising of:
- extracting crude peroxidase from watermelon peel;
- preparing a reaction mixture including the peroxidase extract, guaiacol, hydrogen peroxide, a metallic chloride salt selected from the group consisting of CuCl₂, FeCl₃, KCl, and MgCl₂, and a buffer solution;
- optimizing the concentration of the metallic chloride salt to enhance the enzyme activity;
- wherein the enzymatic activity is determined by monitoring the oxidation of guaiacol using spectrophotometric analysis at 470 nm.
2. The method of claim 1, wherein the concentration of the metallic chloride salt ranges from 0.5 mM to 2 mM.
3. The method of claim 1, wherein the buffer solution is a sodium acetate buffer with a pH of 5.4.
4. The method of claim 1, wherein the optimal concentration for CuCl₂ is 1.5 mM, for FeCl₃ is 1 mM, and for KCl and MgCl₂ is 2 mM.
5. The method of claim 1, wherein the reaction mixture has a total volume of 3 mL.
6. The method of claim 1, wherein the oxidation reaction is initiated by the addition of hydrogen peroxide at a concentration of 2 mM.
7. The method of claim 1, wherein the crude peroxidase extract is obtained by homogenizing watermelon peel, filtering the homogenate, and collecting the filtrate as the enzyme source.
8. The method of claim 1, wherein the enzyme activity is measured by calculating the initial velocity of the reaction from the slope of the absorbance curve recorded at 470 nm.
9. The method of claim 1, further comprising using the activated peroxidase in applications including wastewater treatment, biosensors, or industrial oxidation processes.
10. The method of claim 1, wherein the metallic chloride salts act as activators by interacting with the active site of the enzyme to enhance its catalytic efficiency.

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