Screening And Characterization Of Key Enzyme Of Effecting The Migration Of Fluoride In Euphausia Superba

Antarctic krill contains various nutrient active substances such as protein,amino acid,fatty acid and astaxanthin.It has become a research hotspot around the world.Antarctic krill can enrich fluorine.And its fluorine content can reach 2400mg/kg?dry basis?,being 1000 times of the fluorine content in the ordinary food.The high fluorine content restricts its development and utilization of protein resources.In the paper,the enzyme system in Antarctic krill was analyzed,and the key enzymes influencing the dissolution of fluoride from shrimp shell were selected.On the basis,the key enzymes were purified and their enzymology properties were studied so as to expound fluoride migration mechanism in the process of storage and transportation of Antarctic krill,and provide theory support for the control technology of fluoride.The main conclusions as follows:Enzyme system composition of Euphausia superba was analyzed.The enzyme system composition of Antarctic krill was screened according to International Biochemistry Society of Enzymes recommended enzyme classification.During the range of p H?3.0¹0.0?,the following 10 enzymes were detected,namely,oxidordeuctase?phenol oxidase?,transferase?arginine kinase?,hydrolase?carboxylesterase,cholinesterase,lipase,alkaline phosphatase,pepsin,trypsin and chymotrypsin,carboxypeptidase A?.The enzymes had different activity due to different species.The enzyme activity of lyase,isomerase and synthetase were undetected.Among the 10 enzymes,the activity of phenol oxidase is the highest in oxidoreductase,the specific activity was 12.260±0.935 U/mg.The activity of pepsin is the highest among hydrolytic enzymes,the specific activity was 5.212±0.044 U/mg.The activities of Carboxylate esterase,Cholinesterase,Alkaline phosphatase were lower,the specific activity was below 2.000 U/mg.The maximum enzyme activity of Carboxylate esterase,Cholinesterase,Chymotrypsin,Trypsin was 1.424±0.443 U/mg,1.802±0.907 U/mg,3.970±0.115 U/mg,5.880±0.305 U/mg,the corresponding p H was6.5,7.5,7.5,8.0.The key enzymes affecting the fluoride migration in Antarctic krill were screened.The activity of the corresponding enzymes was inhibited through adding inhibitor so as to screen out the key enzymes affecting the shell fluorine dissolution.The migration content of fluoride was used as the evaluation index,and the significance analysis was carried out.After adding the inhibitor,the inhibition rate of each enzyme was more than 75%.The results showed that the effect of hydrolytic enzymes on the content of fluorine in shrimp shells was significant.The effects of oxidoreductase and hydrolase on the content of fluorine in shrimp shells were not significant.The pepsin,cholinesterase,chymotrypsin,trypsin,carboxypeptidase A,and alkaline phosphatase had significant impacts on the shell fluoride dissolution?P<0.05?.Among them,carboxypeptidase A,chymotrypsin and pepsin were positively correlated with the dissolution of fluorine in shrimp shell,the amount of fluoride migration were above 200 mg/kg.In the ten enzymes,only carboxypeptidase A,chymotrypsin and pepsin were the key enzymes which could influence the fluoride migration in the shrimp shell.At present,More researchfocused on the concern of trypsin in Antarctic krill but studies of carboxypeptidase A are fewer.Trypsin and chymotrypsin were similar in characteristic.Therefore,carboxypeptidase A was selected for the further study.Carboxypeptidase A,the key enzyme influening the fluoride migration in Antarctic krill was separated and purified.Carboxypeptidase A from Antarctic krill was purified by a series of procedures,including ammonium sulfate precipitation,column chromatographies on Hi Trap DEAE FF ion exchange chromatography and SEC 70 gel chromatography.The purification multiple of the protease was 5.12 times,the specific activity was 13.22 U/mg and the yield of the protease was 36.85%.As shown in the result of SDS-PAGE electrophoresis,the molecular weight of this protease was 75k Da.The enzymatic characteristics of carboxypeptidase A in Antarctic krill were studied.The results show that the rate of enzymatic reaction was affected by temperature and p H.Under the reaction conditions of the temperature range of 4⁶0?,carboxypeptidase A had enzyme activity and the optimum temperature was 30?.Under the reaction conditions of p H range of 5¹0,carboxypeptidase A had enzyme activity and the optimum p H was 8.0.Carboxypeptidase A was better stability in the temperature range of 4²0?.Within the p H scope of 7.0⁸.0,carboxypeptidase A showed good stability.The activity of enzyme was more than 90%.Mg²⁺,Zn²⁺,Mn²⁺and Ni²⁺exerted some activation on carboxypeptidase A.The activation effect of Zn²⁺was strongest.Besides,Ca²⁺,Fe³⁺,Cu²⁺and Hg²⁺had inhibitory effect on carboxypeptidase A.The inhibition ability of Hg²⁺was the strongest and the inhibition ability of Fe³⁺was the weakest.Metalloproteinase inhibitors had significant inhibitory effect on carboxypeptidase A,such as EDTA,3-Phenylpropionic acid,1,10-Phenathroline,the inhibitor rate was above 80%.The inhibition rate of TPCK was about 11%,when the concentration of TPCK was10mmol/L.The inhibition rate of aspartic protease inhibitor was less than 5%.