Abstract
Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.
Highlights
Introduction published maps and institutional affilPolyphenol oxidase (PPO) is an oxidase with Cu2+ in its active center [1]
When the tissue is damaged to a certain extent, the massive intervention of oxygen makes the conversion of phenolic substances unbalanced; the quinone in the tissue may cause accumulation; and the quinone substances are further transformed into melanin substances, which leads to the melanization of Acetes chinensis
The unit PPO oxidizes the colorless phenol of Acetes chinensis to colorless bisphenol in the presence of oxygen, and the binary PPO can change the colorless bisphenol to colored quinone [4], which is combined with amino acids in Acetes chinensis to form a brown complex commonly known as black spots [5,6,7]
Summary
Acetes chinensis used in this study was provided by Ruian Huasheng Aquatic Products. (Wenzhou, China), which was harvested from the East China Sea. The body length was about 10–15 mm. Fresh samples were immediately subjected to the extraction of PPO. The other samples were stored in −80 ◦ C ultra-low temperature refrigerator before use
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