Enzymology Properties And Structural Characteristics Analysis On Cold-adapted Trypsin From Antarctic Krill(Euphausia Superba)

Antarctic krill is a key species of crustacean in the South Pole and lives inextremely cold area throughout the year. Euphausia superba has formed a series ofmechanisms to adapt to the extremely cold environment in the process of evolution.And trypsin as the main material of catalytic biological metabolism played a key rolein this mechanism.At present, there have been some related test that cold-adaptedenzymes from other animals have been reported. This article has studied theenzymatic property, fluorescence property and molecular feature of Trypisn fromEuphausia superba aimed to provide data and theory support for future functionalutilization and exploitation.This research was divided into three parts.Part1: Purification and enzymatic characteristics of Trypisn from Euphausiasuperba.The separation and purification of Trypisn-like Enzymes was conducted by crudeextract, DEAE-Sepharose Fast Flow ion-exchange chromatography, and then bySephacryl S100gel chromatography, which used the heads of Euphausia superba.Three kinds of Trypisns were attained and were respectively named S100-Ⅰ,DEAE-Ⅱand DEAE-Ⅲ, the purifity degree were7.463,11.483and12.086respectively. By the method of SDS-PAGE, the molecular weight were about27.16kDa,26.71kDa and26.82kDa orderly. The optimal temperature of enzyme activitywere40℃,45℃and40℃respectively. And with the increase of temperature,enzyme activity decreases gradually. The Kcat of the three enzymes were3.484,9.408and1.816respectively.Part2: Endogenous fluorescence spectrum of trypsin from Euphausia superbaand effects of the benzamidine on the fluorescence quenchingEndogenous fluorescence scanning was carried out on the three kinds ofenzymes, the result shows that the fluorescence peak position of three kinds of trypsins: S100-Ⅰ, DEAE Ⅱ and DEAE-Ⅲ are all near345nm, and there is thetransfer of energy by Tyr no radiation to the Trp; With the increase of temperature,the fluorescence intensity of three kinds of enzyme were low regularity. Quenchingphenomenon by the specificity of the Trypsin inhibitor-benzamidine on enzymeDEAE-Ⅲ shows the weakest. DEAE-Ⅱtook second. S100-Ⅰwas the most obvious.And with the increase of concentration of benzamidine, three kinds of enzymesdisplayed regular lower fluorescence intensity. The quenching of benzamidine on theenzymes of S100-Ⅰand DEAE-Ⅱ belonged to static quenching and there was a seriesof relatively independent binding sites between the benzamidine and two enzymes.Strong interaction between them form the compound. The quenching of benzamidineon the enzymes of DEAE-Ⅲ belonged to combined action of the dynamic and staticquenching. The research on the thermodynamic parameter showed that⊿G of theS100-Ⅰwas the lowest and the⊿H and⊿S were the highest. Also both the catalyticactivity and flexibleness were the strongest but the temperature stability was theweakest. The enzyme of DEAE-Ⅲ had the opposite resμlt. The enzyme of DEAE-Ⅱwas placed in the middle.Part3: The analysis of Euphausia superba on molecular featureAfter extracting the total RNA from trypsin of Euphausia superba, the sequenceof cDNA was acquired by cloning. Then the gene of cDNA and the sequence ofprotein were analysed. The sequences of trypsin from different animals werecompared. The research shows that the number of amino acid of trypsin from animalsdecreased along with evolution and different groups had their own amino acids whichaccount for larger proportion. The molecμlar diameter of trypsin from crustacean wassmaller and had simpler structure but the function was richer. The molecμlar diameterof enzymes from vertebrate was bigger and had more complex structure, but thefunction tends to single. The difference in arginine, lysine, histidine, glutamateaspartic acid, cysteine, alanine, proline, tyrosine and methionine on peripheral andactive center near the site caused significant variation in stability, flexibility andconsumption in reaction as to CAF-TRY, TTF-TRY and CAC-TRY, TTC-TRY,HV-TRY. Meanwhile, their Kcat increased and Km decreased. The catalytic efficiency: CAF-TRY, TTF-TRY>CAC-TRY>TTC-TRY, HV-TRY.