菜籽源锌螯合肽的制备、纯化和结构解析  

Preparation,Purification and Structure Analysis of Rapeseed Source Zinc Chelating Peptide

作　　者：黄晶晶[1] 谢宁宁[1] 李博[2] 程江华[1] 王灼琛[1] 殷俊峰[1] 闫晓明[1] 

机构地区：[1]安徽省农业科学院农产品加工研究所,合肥230031 [2]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《中国粮油学报》2016年第6期68-73,78,共7页Journal of the Chinese Cereals and Oils Association

基　　金：国家科技支撑计划(2012BAD14B13);安徽省农业科学院院长杰出青年基金(14B1211);安徽省农业科学院院长青年创新基金面上项目(14B1253)

摘　　要：采用碱性蛋白酶水解菜籽蛋白制备水解物,运用固定金属亲和层析(IMAC-Zn2+)和葡聚糖凝胶G25(Sephadex G25)层析分离纯化;利用反相高效液相色谱(RP-HPLC)和电喷雾电离质谱(ESI-MS)鉴定序列;采用EDTA络合滴定法(ECT)、双硫腙比色法(DCM)、原子吸收光谱(AAS)和电感耦合等离子体原子发射光谱法(ICP-AES)检测锌螯合率。试验表明:菜籽蛋白的4 h水解物锌螯合潜力最佳;分离该水解物获得3个肽组分(I,Ⅱ和Ⅲ),其中Ⅱ和Ⅲ具有锌螯合能力;进一步分离后分别获得3个(Ⅱ-1,Ⅱ-2,Ⅱ-3)和2个肽组分(Ⅲ-1和Ⅲ-2),组分Ⅱ-3的锌螯合率最高,高于同浓度的谷胱甘肽(GSH)(P<0.05);鉴定Ⅱ-3序列获得4条肽,即Ala-Arg(AR),Asn-Ser-Met(NSM),Gly-Lys-Arg(GKR)和Glu-Pro-SerHis(EPSH)。其中NSM的锌螯合率最高,来源于菜籽蛋白NADH-enoyl-ACP reductase Chain A序列中的296-298氨基酸残基。因此,菜籽蛋白可以作为微量元素螯合肽的优良来源。In this study, rapeseed protein hydrolysates （RPH） obtained from rapeseed meal was hydrolyzed by alcalase and sequentially separated using IMAC -zinc2＋, Sephadex G25 gel filtration and RP- HPLC. The se- quences of chelating peptides were then characterized through ESI -MS. Meanwhile, the zinc -chelating rates were validated by EDTA complexion titration, dithizone chromometer method, atomic absorption spectrometry, and induc- tively coupled plasma atomic emission spectrometry. RPH at 4 h with the highest degree of hydrolysis and zinc - che- lating ability was separated into three peptide fractions （ Ⅰ , Ⅱ, and Ⅲ） after IMAC - zinc2 ＋ What＇s more, the two fractions with zinc chelating abilities （Ⅱ and Ⅲ） were further separated into three fractions （ Ⅱ - 1, Ⅱ- 2, and Ⅱ - 3） and two fractions （ Ⅲ - 1 and Ⅲ - 2 〉, respectively. Among the five fractions, Ⅱ- 3 showed the highest zinc - chelating ability. Moreover, four small peptides, namely, Ala - Arg （ AR）, Asn- Ser - Met （ NSM）, Gly - Lys - Arg （ GKR）, and Glu - Pro - Ser - His （ EPSH）, were obtained and identified from fraction II - 3. NSM, which exhibited the highest zinc - chelating rate, came from the cruciferin cru4 subunit, NADH - enoyl - ACP reduc- tase chain A, and cruciferin precursor of Brassica napus protein. In conclusion, we speculated that utilizing small peptides from rapeseed protein as novel carriers for zinc supplement was feasible.

关 键 词：锌 菜籽蛋白水解物 螯合肽 固定金属亲和层析 碱性蛋白酶 

分 类 号：TS229[轻工技术与工程—粮食、油脂及植物蛋白工程]