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Abstract

Research in Medical & Engineering Sciences

A Comparison of Spectrophotometric and Oxidoreduction Potential Method for Laccase Activity Measurement

  • Open or Close Rana Mustafa1*, Duried Alwazeer2 and B Dave Oomah3

    1Department of Plant Sciences, University of Saskatchewan, Canada

    2Center for Redox Applications in Foods (RCRAF), Iğdır University, Turkey

    3Retired, Formerly with the National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, British Columbia, V0H 1Z0, Canada

    *Corresponding author: Rana Mustafa, College of Agriculture and Bioresources, Department of Plant Sciences, University of Saskatchewan, College of Agriculture and Bio resources, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada

Submission: October 06, 2017; Published: November 07, 2017

DOI: 10.31031/RMES.2017.02.000530

ISSN: 2576-8816
Volume2 Issue1

Abstract

Polyphenol oxidases (PPOs) oxidize phenolic compounds forming quinones, which undergo non-enzymatic reactions resulting in colored compounds. We studied ferulic acid and catechol oxidation by Myceliophthora thermophila laccases using oxidoreduction potential (ORP or Eh) as a novel method to determine PPO activity compared to the traditional spectrophotometric method. Eh varied time-dependently on ferulic acid and catechol concentrations. Eh increased to a maximum value (Ehmax), then decreased irreversibly proportional with substrate concentration. The ΔEh curves of ferulic acid and catechol oxidation at different concentrations by laccase exhibited two steps substrate oxidation: enzymatic and non-enzymatic; linear segments with increasing and decreasing slopes (rate of ΔEh change). The Km values determined spectrophotometrically and ORP were 0.55, 0.75 and 0.39, 0.32mM for catechol and ferulic acid, respectively. The kinetic parameters (Km, Vmax) of enzymatic reaction were not significantly different between the spectrophotometry and ORP methods. The ORP method is simple, low-cost and fast enabling the differentiation between the enzymatic and nonenzymatic phenol oxidation reactions.

Keywords: Myceliophthora thermophila laccases; Polyphenol oxidase; Enzyme kinetic; Oxidoreduction potential; Phenolic compound

Abbreviations: ADA: 4 Amino N, N-Diethyl Aniline Sulphate; KM: Michaels Menten Kinetics Constants; L-DOPA: L-3,4 Dihydroxyphenylalanine; LACs: Laccase; ORP: Oxidoreduction Potential; PPOs: Polyphenol Oxidizes; S: Substrate; V: Reaction velocity; Vmax: Maximum velocity

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