Research On Enzymatic Properties Of Aminopeptidase From Abalone Muscle (Haliotis Discus Hannai) And Its Role In Taste Contribution

Abalone is popular in the market because of its delicate flavor and high nutritive value.Free amino acids have been implicated as major factors characterizing the taste of abalone.During drying process,the content of free amino acids increased,the concentration of several taste-active FAAs such as glutamic acid,glycine,alanine and arginine in dried abalone muscle increased significantly compared with that in fresh abalone muscle.The accumulation of free amino acids is caused by the catalysis of enzymes.Aminopeptidase(AP)is a specific exopeptidase which prefers to remove amino acid residues from the N-terminal of proteins or polypeptides.Peptides and free amino acids which are released by AP are not only nutritious but meaningful to tastes.However,no much information is available regarding the presence of AP in abalone,including its biochemical properties and contribution to the generation of amino acids.Therefore,the present paper aimed to purify an aminopeptidase from abalone muscle and characterize its enzymatic properties.Especially,the relevance of aminopeptidase activity and the variation of free amino acid contents during drying process was investigated,the contribution of aminopeptidases to free amino acids generation in vitro was confirmed.An aminopeptidase was purified to homogeneity from abalone(Haliotis discus hannai)muscle by ammonium sulfate fractionation and column chromatography such as DEAE-Sepharose,Sephacryl S-300,and Phenyl-Sepharose Fast Flow.The molecular weight exhibited was about 100 kDa on SDS-PAGE.Peptide mass fingerprinting analysis obtained 13 peptide fragments containing 147 amino acids which was 100% identical to a puromycin-sensitive aminopeptidase from Crassostrea gigas,which confirmed the purified enzyme is aminopeptidase.The optimum temperature of AP was 30 °C.The activity decreased obviously when the temperature was above 40 °C,suggesting it was not a thermal stable enzyme.The optimum pH of the enzyme was 7.5 and it works in a narrow range of pH from 7.0-8.0,indicating that the enzyme is a neutral aminopeptidase.The purified enzyme only hydrolysed aminopeptidase substrates and showed a broad substrate specificity.Metalloproteinase inhibitors(EDTA,EGTAand 1,10-phenanthrorine)and bestatin could suppress the activity of AP to various degrees.The enzyme lost 70% of the initial activity after completely dialyzed against 1 mmol/L EDTA,whereas the loss could be recovered by Mn2+ and Zn2+,which were proposed to be crucial to maintain the structure of active center and catalyzing.When aminopeptidase was inhibited by bestatin in vitro,the rate of increase in free amino acids was 2.5% after incubation at 30 °C for 2 h,while the control group was 10.6%.These results indicated that aminopeptidases had positive effects on protein degradation and its contribution to the increase of FAA content was noteworthy.The present study also analyzed seasonal variation in the content of taste-active compounds(free amino acids,nucleotide,glycogen)in four species of marine shellfish,including Ruditapes philippinarum,Sinonovacula,Ostrea plicatula,and Haliotis discus hanna.The results indicated that the contents of taste-active amino acids in the four species of marine shellfish were relatively high during the period from January to April,especially in February and March,the content was higher than 2000 mg/kg.However,the content of taste-active amino acids from Ruditapes philippinarum,Haliotis discus hanna,Ostrea plicatula during the period from June to August and that from Sinonovacula in September was relatively low.In terms of taste-active nucleotides,it is abundant in all these four species of shellfish from January to April,especially Sinonovacula and Ruditapes philippinarumrevealed nucleotide content was higher than 400mg/kg.However,the nucleotide content was relatively low between July and October.Meanwhile,the contents of glycogen in four species of shellfish demonstrated similar tendency,they all showed high contents from February to April and low contents in September and October.In conclusion,the contents of taste-active components in the four species ofmarine shellfish were higher in winter and early spring compared to summer and autumn.In conclusion,an aminopeptidase was purified from abalone(Haliotis discus hannai)muscle,the enzymatic properties and its role in the generation of FAAs were discussed.The present study provides a theorical basis to elucidate the function of aminopeptidase in the accumulation of FAA in abalone.In addition,research revealed that the content of taste compounds(taste-active amino acids,nucleotides,glycogen)in marine shellfish is closely associated with seasonal change,which could provide a reference basis for marine shellfish processing.