Purification,Characterization And Molecular Cloning Of Shellfish Paramyosins

Shellfish is an important aquatic animal resource,which has captured the favor of many consumers with its delicious taste and high nutritional value,and also attracted the attention of many researchers.Paramyosin(PM),a structural protein peculiar to invertebrates and particularly abundant(20~40%)in shellfish muscular proteins,plays an important role in the maintenance of muscles texture.Besides,PM was proved to be a major allergen in invertebrates.There were few relevant studies about paramyosin of shellfish at home and abroad so far.In this study,natural paramyosin was isolated from three kinds of shellfish(Haliotis discus hannai,Crassostrea angulata,and Perna viridis),and its basic physicochemical properties were explored.Myofibrillar proteins was obtained from the muscle of Haliotis discus hannai by a washing and salting-in procedures,PM was further purified from this myofibrils with consecutive methods of ammonium sulfate fractionation and hydroxyapatite chromatography.The molecular mass of the PM was about 97 kDa as estimated by SDS-PAGE.The PMs from Crassostrea angulata and Perna viridis were both isolated with the same way that of PM from Haliotis discus hannai.The molecular mass of the PM from Crassostrea angulata was about 97 kDa as estimated by SDS-PAGE.Peptide mass fingerprinting of oyster PM obtained 36 peptide fragments with a total of 403 amino acid residues,which were 100% identical to PM from Crassostres gigas.And the molecular mass of the PM from Perna viridis was about 100 kDa as estimated by SDS-PAGE.Peptide mass fingerprinting of mussel PM obtained 26 peptide fragments with a total of 403 amino acid residues,which were 100% identical to PM from Mytilus galloprovincialis.A polyclonal antibody against PM from abalone muscle was prepared.Western blot showed that polyclonal antibody could specifically reacted with PM from abalone.Besides,the prepared polyclonal antibody could also positively cross reacted with PM from Crassostrea angulata and Perna viridis.The effects of heating on PM were investigated by SDS-PAGE and circular dichroism.The results showed that all three shellfish PMs exhibited good heat resistance.The denaturation temperature(Td)of abalone PM was 52.5±0.2°C,and the denatured secondary structure could be restored after a heating temperature transformation of 80°C to 20°C.The Td of oyster PM was 51.7±0.2°C,and there were about 20% denatured secondary structure could not be restored after a heating temperature transformation of 80°C to 20°C,revealed the worst thermostability in three PMs.The Td of the mussel PM was 56.3±0.2°C,its denatured secondary structure could be restored after a heating temperature transformation of 90°C to 20°C,revealed the best thermostability.The result of SDS-PAGE indicated that the three PMs are relatively stable in the pH range from 6.0 to 11.0,but they were obviously unstable below pH 5.0 and aggregated.The surface hydrophobicity of oyster PM was obviously lower than that of the others.After heating at 100°C for 10 minutes,the surface hydrophobicity of all three PMs increased significantly.In vitro simulated gastrointestinal fluids digestion experiments were carried out to investigate the digestive characterstics of PMs purified from three kinds of shellfish.The results showed that the muscular proteins could not be hydrolyzed completely after continuous digestion by pepsin,trypsin and chymotrypsin.The residual hydrolysates could instantly react with the polyclonal antibody against PM.The three purified PMs showed different digestion patterns in the same conditions of the digestive system,but all showed strong digestion resistance.In addition,the digestion resistance of PMs weakened to some degree after heat treatment.The cDNA of PM with a full-length of 3786 bp was cloned by RT-PCR and RACE,and a accession number of ASU87569.1 was obtained from the GenBank.The complete open reading frame was 2583 bp,encoding a protein composed of 860 amino acid residues,which was 72% and 69% identical to PMs from Mytilus galloprovincialis and Crassostres gigas,showing the high conservation of PMs from molluscs.PM is an important structural protein in shellfish muscle.In this study,we purified PMs from three kinds of shellfish,and explored partial physicochemical properties of the PMs,aiming at providing a valuable theoretical basis for mussel processing and further study on PM.