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该实验建立了HPLC测定大豆胰蛋白酶抑制剂(STI)活性的方法,并对山东产野生大豆(G.soja)与同地区产的黑豆和黄豆(G.max)的胰蛋白酶抑制活性差异进行了比较。用耦合了胰蛋白酶的亲合色谱柱对野生大豆的STI进行分离纯化,紫外分光光度法比较3种大豆的STI含量;PCR结合TA克隆技术对野生大豆STI中的Kunitz型(KSTI)蛋白基因编码区的氨基酸顺序进行初步测定。结果发现,山东产野生大豆的STI活性和含量均高于同地区产的黑豆和黄豆;山东产野生大豆的KSTI蛋白基因编码区的氨基酸顺序与已知的Tia型基本一致,仅第59位氨基酸由于单核苷酸的置换发生了Ser→Thr的转变,此位置位于活性中心附近。研究表明,山东产野生大豆胰蛋白酶抑制活性较强,且含量高。
The experiment established a method for determining the activity of soybean trypsin inhibitor (STI) by HPLC and compared the inhibitory activity of trypsin on the activities of G.soja in Shandong and those of G. max in the same area Compare The STI content of wild soybean was isolated and purified by the affinity chromatography column coupled with trypsin. The STI content of three soybean cultivars was compared by UV spectrophotometry. The Kunitz type (KSTI) protein gene in wild soybean STI was coded by PCR combined with TA cloning The amino acid sequence of the region was initially determined. The results showed that STI activities and contents of wild soybean in Shandong were higher than that of black beans and soybeans produced in the same area. The amino acid sequence of the KSTI protein gene coding region of Shandong wild soybean was basically consistent with the known Tia type. Only amino acid 59 As a single nucleotide substitution occurred Ser? Thr transition, this position is located near the active center. Studies have shown that Shandong wild soybean trypsin inhibitory activity is strong, and high content.