摘要
α-淀粉酶抑制剂(α-amylase inhibitor)是一类能抑制消化道中糖类吸收的物质的统称,它是通过抑制和降低肠道内唾液淀粉酶及胰淀粉酶的活性,阻碍食物中碳水化合物的水解和消化,从而达到减少人体对糖分的摄取,降低人体血液中糖、脂含量的目的。目前,α-淀粉酶抑制剂在医药和农业等领域具有广泛的用途。临床医学上它是防治糖尿病、高血糖、高血脂等病的良好药物;农业上,可将淀粉酶抑制剂基因作为抗虫基因,或者将其制成生物农药;在酶学研究方面,它可作为探究α-淀粉酶活性部位的分析工具或测定α-淀粉酶同工酶的反应物。因此,α-淀粉酶抑制剂应用前景十分广阔。
本实验以白芸豆和小麦麸皮为原料提取分离纯化α-淀粉酶抑制剂,对其工艺条件进行了研究。结果表明:小麦麸皮∶水= 1∶8(100g小麦麸皮,800mL纯净水),浸泡时间2h,添加0.2mol/L的NaCl助溶,并以70%硫酸铵沉淀蛋白,再经超滤膜和冷冻干燥处理,最终得到2.072g粉末,总蛋白含量201mg,比活力为103U/mg,总抑制活力为20720U,收率为41.74%。该抑制剂在pH4-11时较稳定;在80℃下作用30min酶活性丧失10%左右。白芸豆:水=1:8(100g白芸豆,800mL纯净水),24h浸泡过夜,粉碎搅拌4h,添加0.2mol/L的NaCl助溶,得出1.84g干燥样品,配置成浓度为5mg/ml的溶液,比活力为22.2U/mg,总活力为33488U,最终抑制收率为71.9%。该抑制剂在pH3-10时较稳定,80℃下相对稳定。
SDS凝胶电泳测定得出,麸皮α-AI得到三条蛋白质条带,分子量分别是33kD,30kD,26kD;白芸豆α-AI得到两条蛋白质条带,分子量分别为35kD,30kD。对两种α-淀粉酶抑制剂抑制动力学试验表明:小麦麸皮α-淀粉酶抑制剂对α-淀粉酶的抑制类型为混合非竞争性抑制类型,抑制常数为3.47mg/mL;白芸豆α-淀粉酶抑制剂对α-淀粉酶的抑制属于不可逆抑制。
本实验还做了较大规模的小麦麸皮中α-淀粉酶抑制剂的提取,为今后工厂化生产提取提供前期基础数据。
A-amylase inhibitor is a great potential medicine, which can restrain the carbohydrate absorption in the digestive tract. It can effectively inhibit intestinal saliva and pancreatic amylase activity, and impede the hydrolysis and digestion of carbohydrates in food, reduce the intake of sugar, lower blood sugar and blood fat levels. It will not produce a high blood sugar after eating. Thus insulin secretion was reduced and fat synthesis was played down, weight loss. Currently it has a wide range of applications in medicine and agriculture, in clinical medicine it is used to prevent diabetes, high blood glucose, hyperlipemia etc; in agriculture, it also can be used as insect-resistant gene to improve the plant resistance or made of biological pesticides. In addition, in the enzyme research, it can be used as exploreα-amylase site analysis tools, as a determinationα-amylase reagent by selecting process. Therefore, there are very broad application prospects inα-amylase inhibitor.
In this experimentα-amylase inhibitor was extracted from the wheat bran and t white kidney beans,and the technology was researched. The results showed that wheat bran : water = 1:8. The final choice of soaking time was 2h, 0.2mol/L NaCl helped the solution, and 70 percent of the ammonium sulfate precipitation, by the ultrafiltration membrane and freeze-drying processing, and got 2.072g powder sample, the total protein content was 201mg, specific activity was 103U/mg, the total inhibit vitality reached 20720U, recovery was 41.74%. It was stable between pH4-11;under 80 degrees Celsius in 30min, the activity ofα-amylase inhibiter got 10% loss;white kidney bean : water = 1:8. The final choice of soaking time was 24 h, 0.2mol / L NaCl helped the solution, to sulfate precipitation with 1:1 alcohol, eventually got 1.84g powder sample, specific activity was 22.2U / mg, the total inhibit vitality reached 33488U, recovery was 71.9%. It’s stability in pH3-10, it was stable under 80 degrees Celsius.
We got three bands fromα-AI of wheat bran by SDS PAGE. the moleculars were 33kD,30kD and 26kD;two bands were got from white kidney bean, he molecular were 35kD and 30kD. The inhibition kinetics showed the type ofα-AI of wheat bran was mixed non-competitive inhibition,the constant of inhibition (Ki) was 3.47mg/ml. the inhibition of white kidney beanα-AI was non-reversible.
We also maked the experiment of large-scale extraction from wheat bran, gived the basic date for industry production.
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