Characterization Of Polyphenol Oxidase From Shrimp And Development Of A Composite Preservative

Polyphenol oxidase(EC 1.10.3.1, PPO) is an oxidoreductase that its active centre contains copper ion, PPO widely exists in microorganisms、 animals and plants. PPO can react with phenolics, leting phenolics into colorless quionoes, then the quionoes aggregates into melanoma.White shrimp(Litopenaeus vannamei) is one of the three largest farming species, it is widely consumed because of its thin shell and low fat content high mutritional value and delicious taste. However, due to the exsitence of the PPO in the head of shrimp, it is easy to become black and loss nutrients and affect its makert value during storage.In the present study, a PPO was purified from the head of white shrimp by ammonium sulfate fractionation and column chromatographies such as DEAE-Cellulose anion-exchange chromatography, Phenyl-Sepharose HP hydrophobic chromatography, Hi-Trap Capto-Q anion-exchange chromatography. SDS-PAGE and Native-PAGE showed that PPO was highly purified. MALDI-TOF/TOF mass spectrometry was used to confirm the purified protein. Fifteen peptide fragments with a total of 187 amino acid residues were obtanined and revealed 100 % identity to phenol oxidase from white shrimp. Using L-Dopa as substrats, the optimal temperature of PPO was 40 and optimal p H was 6. PPO was stable in the p H range from 5 to ℃9. The enzyme activity of phenol was inhibited EDTA and 1,10-phenathorline, which indicated is an enzyme that needs ions. Additives such as Vc, tea-polyphenols, cysteine, and TBHQ can inhibit the enzyme activity. On the other hend the enzyme lost 64 % of its activity after deal with 5 mmol/L Cu2+ and the enzyme enhance 18% of its activity after deal with 5 mmol/L Mg2+.Through enzyme characterization, a composite of preservative based on 0.1g/L TB-H Q, 0.01g/L 4-hexylres-orcinol(4-HR) 1.5 g/L Vc, 1 g/L tea-polyphenols was develop t-o replace sodium sulfites comparing to use high concentrations of 4-HR(0.02 g/L). It caneffectively delay the melanosis of white shrimp and reduce the total number of colonies,TVB-N and K value during storage. The composite preservative can effectively solve th e problem of melanosis and extend the shelf life of shrimp during stroage. Our research provides new approach for extension the storage period of white shrimp.