Effects of high pressure processing on the polyphenol oxidase activity: exploring the relationship between structure conformation and enzyme activity using Markov State Model

Abstract

Polyphenol oxidase (PPO), a copper-containing enzyme, plays a key role in the enzymatic browning of fruits and vegetables, presenting challenges for food preservation. High-pressure processing (HPP) is a non-thermal technology for preserving fruits, vegetables, meat, etc. In some cases, its effect on PPO activity is limited, leading to severe browning during storage, and the underlying mechanisms remains unclear. This study purified mushroom PPO, primarily with a molecular weight of 50 kDa, and its conformation and activity under different HPP conditions were evaluated by combining multispectral analysis with computational methods. Results showed that enzyme activity decreased with increasing pressure, retaining 16.55%, 13.28%, and 12.76% at 100, 300, and 500 MPa, respectively. High pressure exposed hydrophobic regions of PPO, which promoted ANS binding, led to an increase in fluorescence intensity along with a blue shift. The secondary structure became less stable—β-folding rose by 19.3%, while random coils dropped by 13.3%. As more fluorescent groups were exposed, fluorescence quenching was enhanced. Molecular dynamics simulations combined with Markov State Models (MSMs) revealed that high pressure stabilized PPO, whereas pressure release caused local structural disruptions, increased flexibility, and a looser overall conformation. For MSM analysis, conformational distributions were compact during HPP and showed varied expansion directions depending on the release pressure. High pressure under 100 MPa showed limited conformation changes compared to 0.1 MPa, while under 300 and 500 MPa produced distinct conformations. In addition, after 500 MPa promoted transitions into a unique metastable state not seen under high pressure.

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