纳豆糖蛋白的分离纯化、结构表征及其免疫活性研究

doi:10.7506/spkx1002-6630-201513040

摘要/Abstract

摘要：

为更充分了解纳豆中的成分对脾细胞增殖能力的影响，本实验通过DEAE-52纤维素离子交换柱层析以及Sephadex G-100凝胶柱层析，从纳豆糖蛋白粗提物中获得两种较纯的纳豆糖蛋白：NPPC-1-b和NPPC-2-a。由高效凝胶渗透色谱得到它们的分子质量分别为26.93、357.60 kD。对这两种糖蛋白进行进一步的红外光谱扫描、紫外光谱扫描、单糖组成分析以及氨基酸组成分析显示，NPPC-1-b中含有的是O-糖肽键，而NPPC-2-a中存在的是N-糖肽键。NPPC-1-b主要由甘露糖、鼠李糖、阿拉伯糖组成，且其含量比例分别为67.45%、25.67%和2.44%。NPPC-2-a主要由葡萄糖、甘露糖、阿拉伯糖、木糖以及半乳糖组成，其含量比例分别为36.64%、21.71%、10.35%、9.00%和5.17%。氨基酸自动分析结果显示，两种糖蛋白兼含有人体所需的17 种氨基酸。NPPC-1-b主要由谷氨酸、脯氨酸、赖氨酸和精氨酸组成，其含量分别为0.971%、0.791%、0.708%和0.603%；NPPC-2-a主要由谷氨酸、脯氨酸以及天冬氨酸组成，其含量分别为0.903%、0.689%和0.504%。此外，体外免疫活性探讨发现，100～200 μg/mL的NPPC-1-b以及12.5～200 μg/mL的NPPC-2-a可显著增强脾细胞增殖能力（P＜0.05），其中以100 μg/mL的NPPC-2-a作用效果最佳。在细胞因子表达方面， NPPC-1-b和NPPC-2-a可显著促进细胞因子白细胞介素-2（interleukin-2，IL-2）以及干扰素-γ（interferon-γ，IFN-γ）的表达，其中50 μg/mL的NPPC-2-a分泌IL-2最多，200 μg/mL的NPPC-2-a表达IFN-γ的量最高。以上研究结果表明NPPC-1-b和NPPC-2-a具有一定的免疫调节作用。

关键词: 纳豆, 糖蛋白复合物, 结构分析, 免疫活性

Abstract:

To fully understand the proliferation of splenocyte in vitro of natto ingredients, two kinds of polysaccharideprotein
complexes, named as NPPC-1-b and NPPC-2-a, were extracted and purified from natto by DEAE-52 cellulose ionexchange
chromatography and Sephadex G-100 gel filtration chromatography. Their molecular weights were determined
by performance gel permeation chromatography to be approximately 26.93 and 357.60 kD for NPPC-1-b and NPPC-2-a,
respectively. The two complexes were further characterized by FT-IR, UV-visible spectrophotometry, monosaccharide
and amino acid composition analysis. The results showed that NPPC-1-b contained an O-glycopeptide bond, while
NPPC-2-a contained an N-glycopeptide bond. NPPC-1-b was mainly composed of mannose (Man), rhamnose (Rha)
and arabinose (Ara), with the contents of 67.45%, 25.67% and 2.44%, respectively. However, NPPC-2-a was mainly
composed of glucose (Glc), mannose (Man), arabinose (Ara), xylose (Xyl) and galactose (Gal), accounting for 36.64%,
21.71%, 10.35%, 9.00% and 5.17%, respectively. Seventeen amino acids were identified in both complexes. NPPC-1-b
was mainly composed of glutamic (Glu), proline (Pro), lysine (Lys) and arginine (Arg) with contents of 0.971%, 0.791%,
0.708% and 0.603%, while NPPC-2-a was mainly composed of Glu, Pro and aspartic (Asp) acid with contents of 0.903%,
0.689% and 0.504%. Besides, preliminary in vitro tests showed that NPPC-1-b (100–200 μg/mL) and NPPC-2-a (12.5–
200 μg/mL) exhibited significantly higher immunomodulatory activities against the proliferation of lymphocytes
(P < 0.05), and the effect of 100 μg/mL NPPC-2-a was the best. In respect of cytokines expression, NPPC-1-b and NPPC-2-a
significantly enhanced the expression of interleukin-2 (IL-2) and interferon-γ (INF-γ), and 50 μg/mL NPPC-1-b showed a
strong effect on IL-2 secretion, 200 μg/mL NPPC-2-a exhibited a strong effect on INF-γ secretion. These results suggest that
NPPC-1-b and NPPC-2-a can effectively increase immunomodulatory activity.

Key words: natto, polysaccharide-protein complexes, structure analysis, immunomodulatory activity

中图分类号:

沈柱英，黄占旺*，肖建辉，曹靖文. 纳豆糖蛋白的分离纯化、结构表征及其免疫活性研究[J]. 食品科学, doi: 10.7506/spkx1002-6630-201513040.

SHEN Zhuying, HUANG Zhanwang*, XIAO Jianhui, CAO Jingwen. Purification, Structural Characterization and Immunomodulatory Activity of Natto Polysaccharide-Protein Complexes[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201513040.

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纳豆糖蛋白的分离纯化、结构表征及其免疫活性研究

Purification, Structural Characterization and Immunomodulatory Activity of Natto Polysaccharide-Protein Complexes

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