白芸豆中α-淀粉酶抑制剂糖蛋白的提取纯化、组成结构及生物活性研究

【作者】 杨明琰；

【导师】 宋纪蓉；

【作者基本信息】 西北大学 ， 化学工程， 2008， 博士

【摘要】 本文采用现代分离纯化技术从白芸豆中分离提取得到单一组分的α-淀粉酶抑制剂(α-AI)糖蛋白,对其生物学性质、超滤提取工艺、生物降糖、降脂活性及结构与功能之间的构效关系进行了系统的研究。采用柱色谱分离技术从白芸豆中分离得到单一组分的α-AI,其为相对分子量为36000 Da的糖蛋白,对猪胰α-淀粉酶具有较高的抑制活性,抑制类型呈非竞争性抑制。白芸豆α-AI糖蛋白对温度、pH具有较高的稳定性。白芸豆α-AI糖蛋白中蛋白质含量占88.2%,蛋白质中含有17种常见氨基酸,其中天冬氨酸、谷氨酸、丝氨酸和苏氨酸占有较高的比例;糖链中单糖组成为甘露糖、葡萄糖、半乳糖和木糖,其摩尔比为2.42:1.50:1.52:1.00,糖链中单糖残基以α-糖苷键连接。糖蛋白中蛋白质和糖链的连接方式为O-糖肽键。构效关系研究结果表明α-AI糖蛋白中单独的糖链和蛋白链都没有生物活性,只有糖链和蛋白链结合在一起形成完整的糖蛋白时,才对猪胰α-淀粉酶具有抑制活性。分别采用有机膜超滤和无机膜超滤对白芸豆提取液中α-AI糖蛋白的浓缩提取工艺进行了对比研究。结果表明截留分子量为10000 Da的有机超滤膜更适合白芸豆中α-AI糖蛋白的分离浓缩。其最佳工艺条件为操作压力0.8MPa,物料质量分数7%(1:15),物料温度35℃,物料pH为自然pH。在此条件下,其稳定膜通量为35.6 L/m~2h,体积浓缩倍数为8.3倍,多糖的截留率为20.1%,蛋白质的截留率为72.7%,活性物质收率为80.6%。对白芸豆中α-AI的食用安全性和生物降糖、降脂活性进行了研究。结果表明α-AI无急性毒性;α-AI能显著降低四氧嘧啶糖尿病大鼠的空腹血糖,降低餐后血糖峰值,增加其淀粉耐量;α-AI能显著降低高血脂模型大鼠血清的TG、TC和LDL-C含量,升高HDL-C含量,具有调节高血脂模型大鼠血脂的功效。更多还原

【Abstract】 A homogeneous glycoproteinα-amylase inhibitor (α-AI) was extracted and purified from white kidney beans (Phaseolus vulgaris. L) by a series of modern technologies. The biochemical characterization, ultrafiltration technology , hypoglycemic, hypolipidemic activity and structure/function relationship were systematically studied in this dissertation.Anα-AI was purified from white kidney beans (Phaseolus vulgaris. L) by a series of chromatography. It was a 36000Da homogeneous glycoprotein and was effective against porcine pancreatic amylase and presented as an uncompetitive inhibition. The activity ofα-AI was stable to pH and temperature.Theα-AI glycoprotein contained 88.2% protein and was rich in aspartic acid, glutamic acid, serine and threonine. The carbohydrate moiety consisted of Man, Glc, Gal and Xyl in a mole ratio of 2.42:1.50:1.52:1.00. The sugar chain was connected byαglucosidic bond. The glycan and the core protein backbone were connected by O-linkage.The study of relationship between structure and function showed there was no inhibitory activity when using protein or sugar chain along; only when protein and sugar chain were linked together, it presented inhibitory activity.Compared with inorganic ceramic membrane ultrafiltration, Organic membrane ultrafiltration was more suitable for concentratingα-AI glycoprotein from solution of white kidney beans. The optimum parameters were as follows: retentive molecule 10000Da, pressure 0.8 MPa, solution concentration 7%(1:15), temperature 35℃; natural pH. In this condition, the flux of membrane was 35.6 L/m~2h, the volume was concentrated 8.3 times, retentive ratio of polysaccharide was 20.1%, retentive ratio of protein was 72.7%, extracting ratio ofα-AI was 80.6%.The study of acute toxicity, hypoglycemic and hypolipidemic activity ofα-AI from white kidney beans were as follows:α-AI was nontoxic;α-AI could significantly reduce fast blood sugar, decrease the postprandial rise in blood concentration and improve sugar tolerance in alloxan-dependent diabetic model rats;α-AI could significantly reduce the concentration of TG, TC, LDL-C and increase the concentration of HDL-C in serum of hyperlipidemia model rats.更多还原