大豆胰蛋白酶抑制剂的制备及性质

Preparation and Properties of Soybean Trypsin Inhibitor

采用硫酸钠盐析法从大豆乳清废水中选择性回收大豆胰蛋白酶抑制剂(soybean trypsin inhibitor,STI),且以商品化的Kunitz型胰蛋白酶抑制剂(soybean Kunitz trypsin inhibitor,KTI)为对照表征STI的理化性质和界面性质。结果表明,STI提取优化条件为:乳清溶液固形物含量13%、pH 4、加盐量9 g/100 m L,此条件下STI的得率为20.54%;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示,其主要成分为KTI,以苯甲酰-DL-精氨酸-p-硝基酰替苯胺盐酸盐为底物的胰蛋白酶抑制活力为2 135.00 TIU/mg,且具有良好的温度和pH值稳定性(80℃加热30 min后仍保持73.19%的抑制活力,在pH 2~11范围内抑制活力无明显变化);傅里叶变换红外光谱和圆二色性结果显示,其与KTI(Sigma T9218)的结构类似,二级结构主要是β-折叠和无规卷曲;界面性质数据表明,STI分子能很快吸附到气水界面形成高弹性界面,从而使其具有良好的起泡性和泡沫稳定性。因此,简单的硫酸钠盐析法是大规模制备高纯度且功能性质良好的STI的有效方法,所获得的STI在医药及功能性食品领域有潜在的应用价值。

Sodium sulfate salting out was employed to selectively recover soybean trypsin inhibitor (STI) from soybean whey waste. The recovery conditions were optimized, and the physicochemical and interfacial properties of STI were evaluated. The results indicated that the yield of STI was 20.54% under optimal conditions: concentration of soybean whey to a solid content of 13%, adjustment to pH 4, and addition of 9 g/100 mL sodium sulfate. The main component of STI was Kunitz trypsin inhibitor (KTI, 20.1 kD) as indicated by SDS-PAGE analysis. The trypsin inhibitor activity towards Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA) of STI was 2 135.00 TIU/mg at pH 7. It was stable in a broad pH range from 2.0 to 11.0 and at a temperature up to 80 ℃. The inhibitory activity (73.19%) was still maintained at a high level after heating at 80 ℃ for 30 min. The FTIR and CD spectra showed that the structure of STI was highly similar to that of the commercial KTI (Sigma T9128). The dynamic surface tension and surface dilatational parameters of STI showed that the STI molecules could be quickly adsorbed to the interface to form a high elastic network and hence favorable foaming capacity and foaming stability. These findings suggest that the salting out method could be used as an effective strategy to prepare high-purity STI from soybean whey, and STI would emerge as a promising molecule for functional foods and medical applications because it exhibits excellent temperature and pH stability and high bioactivity.