Affinity chromatography studies for mespilus germanica L. polyphenol oxidase

Area of Science:

• Food Science
• Enzymology
• Biochemistry

Background:

• Polyphenol oxidase (PPO) enzymes catalyze enzymatic browning in foods.
• Medlar fruit (Mespilus germanica L.) is a valuable food source with antioxidant and antiviral properties.
• Efficient purification of PPO is crucial for understanding its role in food quality and potential applications.

Purpose of the Study:

• To purify and characterize polyphenol oxidase (PPO) from medlar fruit.
• To develop and apply novel affinity chromatography methods for PPO purification.
• To determine the kinetic properties and subunit structure of medlar PPO.

Main Methods:

• Partial purification of PPO using homogenization and ammonium sulfate precipitation.
• Enzyme characterization including optimal substrate, buffer, pH, and temperature determination.
• Purification of PPO using Sepharose 4B-L-tyrosine-p-aminobenzoic acid (S-4B-TABA) and Sepharose 6B-L-tyrosine-p-aminobenzoic acid (S-6B-TABA) affinity gels.
• Analysis of enzyme purity and subunit structure using Native PAGE and SDS-PAGE.

Main Results:

• Optimal conditions for medlar PPO activity with catechol were determined as 0.1 M, pH 6.8, and 15 °C.
• Kinetic parameters Vmax and KM for catechol were calculated as 12,542.46 IU and 2.5 mM, respectively.
• High purification degrees (54.0 for S-4B-TABA, 4.8 for S-6B-TABA) were achieved.
• Native PAGE and SDS-PAGE revealed a single subunit of 40 kDa, indicating a homogenous enzyme preparation.

Conclusions:

• Medlar PPO was successfully purified for the first time using S-4B-TABA and S-6B-TABA affinity chromatography.
• This study establishes a novel and effective method for purifying medlar PPO.
• The characterized medlar PPO provides valuable insights for food industry applications and further enzymatic research.