南极磷虾酶解产物中一种含氟多肽的研究

通过酶解法从南极磷虾肌肉酶解产物中分离、纯化含氟多肽,并采用LC-MS/MS和氨基酸序列分析对其结构进行研究。结果表明,采用胰蛋白酶酶解所得南极磷虾多肽粗提物的得率约为20%,其总氟含量为(731.73±1.01)μg·g^-1。粗提物精制后,制备得到纯度较高的6个多肽(分别命名为F1、F2、F3、F4、F5、F6),采用离子色谱法筛选出含氟量最高的多肽F6,测定其结合肽氟含量为(266.11±0.40)μg·g^-1。将F6的氨基酸序列片段通过NCBI-Nucleotide-Euphausiacea蛋白质数据库检索,按匹配值得分高于100分可筛选鉴定得到4种蛋白质Clock,Tropomyosin,ATP synthase subunit 6(mitochondrion),Arginine kinase],通过对4种蛋白的分析发现,南极磷虾蛋白结合氟通过与Clock蛋白磷酸化结合的可能性最大。研究结果对今后探究南极磷虾中含氟蛋白的安全评价提供了技术支撑,对南极磷虾应用价值的进一步开发具有重要意义。

Study on a fluoropeptide in enzymatic hydrolysis products of Antarctic krill

Antarctic krill,which is rich in polyunsaturated fatty acids,proteins,minerals,and a large quantify of vitamins A and E,possesses the potential to be harvested with 379 million tons of total biomass and 342~536 million tons of annual gross post-larval production.However,Antarctic krill contains a high amount of fluoride in different tissues.Although fluoride is helpful in prevention of dental caries and strengthening bones in moderate contents,it can also cause fluorosis disease if existing with an excessive amount in man and animals.As WHO recommended 2~4 mg and 1~2 mg of daily intake of fluoride for adults and children respectively,Antarctic krill is not acceptable for direct consumption as food.In this study,the fluorinated polypeptide was released through enzymatic hydrolysis.The hydrolysate of Antarctic krill was isolated through ultrafiltration with molecule cutoff filter.The filtrate was lyophilized to get the powder.Furthermore,the powder was fractioned and separated with GPC and HPLC.Its fluoride content was further determined through ion chromatography after hydrolyzing these polypeptides with hydrogen chloride.The polypeptide with highest fluoride content was measured by LC-MS/MS and analyzed through amino acid sequence analysis.Moreover,after analysis and search for homologous protein through NCBI database,the most related protein was identified and discussed.The experimental results showed that the crude extract of Antarctic krill peptide obtained by trypsin digestion was 20%,and the total fluorine content was(731.73±1.01)μg·g^-1.The crude extracts were repeatedly refined by Sephadex G-25 Sephadex chromatography and C18 reverse-phase column.Six peptides with higher purity(named F1,F2,F3,F4,F5,F6,respectively)were obtained.F6 polypeptide was found to contain the highest fluorine through the analysis with ion chromatography after acid hydrolysis for fluoride release.The fluorine content of the bound peptide(F6)was determined to be(266.11±0.40)μg·g^-1.The least fluoride content of the bound polypeptides was determined to be(25.42±0.69)μg·g^-1.Furthermore,LC-MS/MS was used to identify the amino acid sequence fragment of the polypeptide F6.Through the NCBI-Nucleotide-Euphausiacea protein database search,four proteinsClock,Tropomyosin,ATP synthase subunit 6(mitochondrion),Arginine kinase]were identified with match scores above 100.Analysis of the four proteins revealed that F6 was most likely related to Clock proteins through phosphorylation of the bound fluorine in the Antarctic krill protein.The results will provide technical support for the future safety evaluation of fluoride-containing proteins in Antarctic krill,and are of great significance for the further development of the application value of Antarctic krill.