Molecular Cloning,Expression And Antibody Preparation Of Polyphenol Oxidase From Litopenaeus Vannamei

Blackening（i.e.,melanosis）is a phenomenon of black spots at the junction of the crustacean carapace,caused by a series of automatic oxidation and polymerization reactions.Melanosis is a typical feature of shrimp causing quality decrement after death.Polyphenol oxidase（PPO）is a critical enzyme responsible for the melanosis of shrimp after death,which leads to melanosis and the reduction of sensory quality of shrimp.Therefore,investigation of the properties of PPO will help to reveal its enzymatic characteristics.This study has theoretical implications and potential practical value for the development of PPO-specific inhibitor to retard melanosis in shrimps.However,natural PPOs are low in content and poor in stability,causing it quite difficult to purify and investigate such enzymes in depth.In this study,a typical shrimp species,Litopenaeus vannamei was used to clone the PPO gene（Lv-PPO）using molecular biology.The Lv-PPO gene was further expressed in vitro and purified to homogeneity.The basic enzymatic properties of recombinant Lv-PPO（r Lv-PPO）were investigated in detail.A specific polyclonal antibody against r Lv-PPO was prepared.The tissue distribution of PPO was detected.The function of PPO in the melanosis process of shrimp was analyzed with a purpose to provide insight into the function of PPO in the melanosis process of shrimp and reveal the mechanism of PPO in shrimp melanosis.Lv-PPO was cloned and expressed by constructing the Escherichia coli prokaryotic expression system and the expressed protein was then purified by affinity column chromatography.The Lv-PPO gene is 2076 bp in length,encoding 691 amino acid residues with a presumed molecular weight of 78.8 k Da.r Lv-PPO was mainly expressed in the form of inclusion body,partially soluble,with a molecular weight of approximately 85 k Da.The expressed protein was confirmed by LC-MS/MS to be r Lv-PPO.r Lv-PPO was biologically active with an optimal temperature of 40℃and an optimal p H of 6.0.Its denaturation temperature was 54.2±0.7°C as obtained by circular dichroism.As a metalloenzyme,the enzymatic activity of r Lv-PPO was inhibited by metal chelators such as EGTA and ETA,while acids can also reduce the enzymatic activity of r Lv-PPO.Metal ions Cu²⁺and Zn²⁺revealed a low concentration promoting and high concentration inhibiting effect on the enzymatic activity of r Lv-PPO,while high concentration of Co²⁺also able to inhibit the enzymatic activity of r Lv-PPO.Further studies revealed that Cu²⁺and Zn²⁺altered the activity of r Lv-PPO by affecting its secondary and tertiary structure.r Lv-PPO showed strong catalytic activity towards the redox reactions mediated by the substrates L-Dopa and catechol with their Michaelis constant K_m values of 1.84 mmol/L and 0.92 mmol/L,respectively.r Lv-PPO exists in live shrimps in the form of zymogen（pro PPO）,which requires activation by serine protease cascade reaction.Surfactant SDS is able to activate pro PPO in vitro.To resolve the presence of PPO in L.vannamei at the protein level,a specific polyclonal antibody against r Lv-PPO was prepared.The distribution of PPO in different tissues of L.vannamei was investigated by Western blot in vitro.The result showed that the highest level of PPO was found in the hemolymph,followed by the tail,carapace and eye stalk of the shrimp.Only small amount of PPO was detected in the muscle,while the presence of PPO was not detected in the hepatopancreas and midgut,and the result was generally consistent with the appearance of melanosis in shrimp.As an economically important aquatic animal,the economic loss caused by post-mortem melanosis of shrimp is enormous.Inhibition of PPO activity is the key to prevent and control post-mortem melanosis in shrimp.In this study,the biologically active r Lv-PPO was expressed for the first time and a specific polyclonal antibody was prepared to against it,which can help to gain insight into the enzymatic properties of r Lv-PPO and provide an effective means to explore the mechanism of PPO in shrimp melanosis.