Purification,Enzymatic Properties And Mechanism Of Cold-Adaption Of Carboxypeptidase From Euphausia Superb

Carboxypeptidase is a protease,which can hydrolyze amino acid residues from the c-terminal of peptide chain.It was widely found in higherplants,animal and fungi.At present,relevant studies have involved the separation and purification,enzymatic properties,molecular structure characteristics and gene recombination of enzymes,but most of the research objects were focused on microorganisms,and relatively few of them have special cold-adapted characteristics for animals,especially the Euphausia superb Therefore,a carboxypeptidase was isolated and purified from E.superb,and its enzymatic properties were studied.Based on the whole gene sequence of E.superb obtained by transcriptome sequencing in the early stage of the research group,the structure of carboxypeptidase was predicted by bioinformatics method through the gene transfer of carboxypeptidase,so as to reveal its high-efficiency catalysis in extremely cold environment The mechanism of energy provides a reference for its molecular modification and application in food and bioengineering.Carboxypeptidase from E.superb was extracted first with buffer solution and then purified respectively with ammonium sulfate,DEAE-agarose gel FF anion chromatography and Sephadex G-100 gel chromatography.The results of SDS-PAGE showed its molecular weight was at about 30 ku.Aspects such as optimal temperature,optimal p H,thermal stability,activations and inhibitions allowed an in-depth understanding of the enzymatic properties of arboxypeptidase from E.superb.Its optimal temperature was at 30°C and optimal p H was at 8.0.The low thermal stability resulted in a significant diminishing of enzyme vitality after two hours of autolysis,even at 4°C.The metal ions Ni²⁺,Mn²⁺,Zn²⁺,Mg²⁺ had the activation effect.When the concentration was 1mmol/L,the activation effect of Zn²⁺ was more significant;when the concentration was 5mmol/L,the activation effect of Mn²⁺ was more significant;when the concentration is 10mmol/L,the activation effect of Mg²⁺ was more significant.Ca²⁺?Fe3+ and Cu²⁺ inhibited its enzymatic activity and Cu²⁺ was with the strongest inhibitory.DTT and ?-mercaptoethanol,inhibitors of thiol modification,showed inhibitory on the carboxypeptidase.It was indicated that there were disulfide bonds in its active center.Metalloproteinase inhibitors EDTA and 1,10-phenanthroline had significant inhibitory on its enzymatic activity,which was similar with the characteristics of metalloproteinase.While Serine protease inhibitor PMSF didn't show significant effect on its activity.The kinetic constants of Km and Vmax were revealed at 0.0059mg/m L and 4.9091U/min respectively with hippuryl-L-phenylalanine as a substrate.In this study,we obtained two complete gene information of carboxypeptidase of E.superb,the total length of c DNA is 1070 bp,the largest ORF region encodes 301 amino acid residues of carboxypeptidase CP1 of E.superb;the largest ORF region encodes 562 amino acid residues of carboxypeptidase CP2 of E.superb.The two gene sequence information has been submitted to Gen Bank with the login numbers of MK696972 and MK96973 respectively.Based on the kinetic comparison of carboxypeptidase and bovine carboxypeptidase of E.superb,the structure and chilling adaptability of carboxypeptidase of E.superb were studied by bioinformatics,physicochemical properties,amino acid sequence comparison and secondary and tertiary structure prediction.The cold-adaption of carboxypeptidase from E.superb was initially examined by comparing its thermodynamic activation parameters with its counterparts from bovine.Then the cold-adaption mechanism was discussed with the full gene and predicted structure based on bioinformatics.The results showed the catalytic efficiency Kcat/Km and affinity to substrate of E.superb carboxypeptidase were higher than its counterparts of bovine at 4°C and 30°C respectively and revealed its cold-adaption behavior.The amino acid sequence of E.superb carboxypeptidase was highly homologous with many species,such as Astacus astacus,as seen in the result of sequence alignment.The active site and Zn binding site were also proved with conservation.Its cold-adaption may be attributed to the following attributes.The proportions of certain residues were critical in cold-adaptation behavior,such as a higher proportion of Asp and lower proportions of Pro,Arg and Phe comparing with its warm-counterparts.It could probably diminish the number of intra molecular interaction resulting in improved structural flexibility.Furthermore,higher proportion of loose random coils and reduced steric hindrance might be also the key factors promoting its cold-adaption.Based on the conservation region analysis of carboxypeptidase CP2 of E.superb,it has strict conservation with the carboxypeptidase sequence of all comparative species in the Zn binding site,the active site of catalytic center and other regions.CP2 amino acid sequence and Penaeus vannamei,Eurytemora affinis,Crassostrea compared with other normal temperature animals,CP2 has a lower ratio of pro,Tyr,Arg and Lys,and the secondary and tertiary structure of CP2 was predicted.The results showed that the secondary component of CP2 was mainly composed of ?-helix and irregular crimp,and the proportion of irregular crimp is relatively high.