Isolation and Partial Purification of Polyphenol Oxidase from Banana for Biosensor Applications

Abstract

An amperometric biosensor based on the enzyme polyphenol oxidase (PPO), isolated and partially purified from banana, was developed using catechol as substrate. Co-crosslinking method of immobilization was adopted using the protein-based stabilizing agent, bovine serum albumin (BSA) on cellophane and nylon membranes with glutaraldehyde as the crosslinking agent. The parameters measured were the sensitivity, the concentration range of the linear dependence of the sensor response to catechol. The enzyme electrode gave detection limit of 10 x10-5 M and 5 x10-5 M and linear response range of 20-80 x10-5 M and 10-60 x10-5 M for catechol employing cellophane as well as nylon membrane respectively. Biosensor response reached steady state within 3 min for both cellophane and nylon membranes and exhibited maximum activity at 25 0C and pH 6.0.

Introduction

• Phenolic compounds are common in chemical, petrochemical, and pharmaceutical industries, but pose serious health hazards when released into water.

• Some phenols (like catechol derivatives) also occur naturally in human physiology, making their trace-level detection important in both environmental and clinical settings.

• Conventional detection methods (HPLC, GC, spectrophotometry, etc.) are expensive, time-consuming, and not suitable for continuous monitoring.

• Enzyme-based amperometric biosensors offer a cost-effective, sensitive, and selective alternative, often using enzymes like tyrosinase, laccase, and horseradish peroxidase (HRP).

Objective

To develop an amperometric biosensor using polyphenol oxidase (PPO) extracted from banana, immobilized via co-crosslinking with BSA and glutaraldehyde on cellophane and nylon membranes, for phenol detection (specifically catechol).

Materials and Methods

• PPO Extraction: Banana pulp was processed, and PPO was extracted using phosphate buffer, followed by ammonium sulfate precipitation and dialysis.

• Enzyme Assay: Spectrophotometric method at 420 nm with catechol as substrate.

• pH and Temperature Studies:

  • Optimum pH: ~5.0; enzyme activity dropped above pH 6 due to possible conformational changes or side reactions.

  • Optimum Temperature: 25°C.

• Biosensor Assembly:

  • Enzyme immobilized on membranes using BSA + glutaraldehyde.

  • Immobilized membranes were fixed to a Clark-type oxygen electrode, and measurements were taken via a lab-developed amperometric detector.

Results and Discussion

Enzyme Characterization

• PPO showed maximum activity at 85% ammonium sulfate saturation.

• Stable activity observed at pH 5 and 25°C.

Biosensor Performance

• Linearity observed for catechol concentration:

  • Cellophane: 20–80 × 10?? M

  • Nylon: 10–60 × 10?? M

• Limit of Detection (LOD):

  • Cellophane: 10 × 10?? M

  • Nylon: 5 × 10?? M

• Regression Values:

  • Cellophane: 0.994 (better linearity)

  • Nylon: 0.974

• Performance Comparison:

  • Cellophane: Better linearity and broader detection range

  • Nylon: Better sensitivity and faster response time due to thinner membrane (lower diffusion resistance)

• Conclusion: Cellophane preferred for accurate measurement; nylon better for high sensitivity.

Conclusion

Phenolic compounds have been recognized as toxic substances. Therefore, the determination of phenolic compounds in environmental matrices, including tap and surface water, has become a matter of great concern and scientific interest. Recent research activity has focused on the design and construction of biosensors which are capable of improving the efficiency of site monitoring and can be used for the necessary remediation activities. Polyphenol oxidase-based biosensor reported in this work provides a very good alternative to conventional methods such as HPLC, GC and spectrophotometric techniques with are laborious and time consuming. Banana PPO based biosensor for detection of catechol was developed and biosensor activity was compared on cellophane and nylon membrane support. Results showed that co-crosslinking method of immobilization using BSA on cellophane membrane was considered superior to nylon membrane for the accurate measurement of phenols (R2 = 0.994). Even though nylon enabled lower detection limits for catechol, the accuracy for detection was less (R2 = 0.974). Higher permeability of nylon membrane is the reason for better sensitivity when nylon membrane is used. The banana PPO sensor showed very small response time and we could successfully apply the biosensor in detecting catechol in real water samples.

References

[1] B. R. Albuquerque, S. A. Heleno, M. B. P. S. Oliveira, L. Barros, and I. C F R Ferreira, “Phenolic compounds: Current industrial applications, limitations and future challenges,” Food Funct., vol. 12, pp.14–29, 2021. [2] K. Krol, M. Gantner, A. Tatarak, and E. Hallmann, “The content of polyphenols in coffee beans as roasting, origin and storage effect,” Eur. Food Res. Technol., vol. 246, pp. 33–39, 2020. [3] J. Sanchez-Avila, M. Fernandez-Sanjuan, J. Vincente, and S. Lacorte, “Development of a multi-residue method for the determination of organic micropollutants in water, sediment and mussels using gas chromatography-tandem mass spectrometry,” J. Chromatogr. A, vol. 1218, pp. 6799-6811, 2011. [4] K. Rekha, M. S. Thakur, and N. G. Karanth. “Biosensors for the detection of organophosphorous pesticide,” Crit. Rev. Biotechnol, vol. 20, pp. 213-35, 2000 [5] M. M. Rodriguez-Delgadoa, G. S. Alemán-Navaa, J. M. Rodriguez-Delgadob, G. Dieck-Assadb, S. O. Martínez-Chapab, D. Barceloc, and R. Parraa, “Laccase-based biosensors for detection of phenolic compounds,” TrAC, vol. 74, pp. 21-45, 2015 [6] I. Gul, M. Sheeraz Ahmad, S. M. Saqlan Naqvi, A. Hussain, R. Wali, A. A. Farooqi, and I. Ahmed, “Polyphenol oxidase (PPO) based biosensors for detection of phenolic compounds: A Review,” J. appl. Biol, vol. 5, pp. 72-85, 2017 [7] C. A. Signori Perone, and T. R. L. C. Paixao, “Development and performance evaluation of low-cost cellophane paper-based biosensors for polyphenol detection in teas: A cost-effective alternative to teflon® membranes biosensors,” BrJAC, vol. 11, pp. 64-77, 2024 [8] C. Sarika, K. Rekha, and B. Narasimha Murthy, “Studies on enhancing operational stability of a reusable laccase-based biosensor probe for detection of ortho-substituted phenolic derivatives,” 3 Biotech, vol. 5, pp. 911–924, 2015. [9] M. Y. Cosetang, and C. Y. Lee, “Changes in apple polyphenol oxidase and polyphenol concentrations in relation to degree of browning,” J. Food Sci., vol. 52, pp. 985-989, 1987 [10] M. M. Bradford, “A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding,” Anal. Biochem., vol. 72, pp. 248-254, 1976. [11] T. Zor, and Z. Selinger, “Linearization of the Bradford protein assay increases its sensitivity: Theoretical and experimental studies,” Anal. Biochem., vol. 236, pp. 302-308, 1996.

Copyright

Copyright © 2025 C. Sarika, K. Rekha, B. Narasimha Murthy. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.