灵芝发酵液中蛋白酶抑制剂GLPIA2的纯化及其特性

采用乙醇分级沉淀、凝胶色谱纯化、阴离子交换色谱分离等步骤从灵芝深层发酵液中提取得到蛋白酶抑制剂GLPIA1与GLPIA2。其中GLPIA2仅在215 nm处有紫外吸收,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定为单一条带,相对分子质量为15000。由其氨基酸组成分析谱图可看出,其酸性氨基酸含量较高,碱性氨基酸及芳香族氨基酸含量较低。GLPIA2抑制剂的底物特异性研究表明,它对天冬氨酸族的胃蛋白酶和酵母蛋白酶A有相对较强的抑制作用。

Purification and Characteristic of Proteinase Inhibitor GLPIA2 from Ganoderma lucidum by Submerged

A proteinase inhibitor GLPIA2 was purified to homogeneity from Ganoderma lucidum by submerged fermentation. The purification was carried out by ethanol fractional precipitation (30%-80%), gel-filtration on Superdex 200 column (30 cm x 1.1 cm i.d.) and anion exchange on Source 30Q column (10 cm x 1.6 cm i.d.). The gel chromatographic conditions were as follows: 50 mmol/L sodium phosphate as mobile phase with a flow rate of 1 mL/min with the effluent collection of 1 mL/tube and detection at 280 nm. The anion exchange chromatographic conditions were as follows: 50 mmol/L Tris-HCl (pH 8.8) containing different amounts of NaCl as mobile phase with a flow rate of 2 mL/min with the effluent collection of 5 mL/tube and detection at both 215 nm and 280 nm. Two active fractions named GLPIA1 and GLPIA2 corresponding to proteinase A inhibitory activities were pooled and lyophilized. GLPIA2 only has the absorption at 215 nm. The relative molecular mass of the inhibitor was 15,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of GLPIA2 was analyzed by high performance liquid chromatography. The chromatographic conditions were as follows: C18 column (125 mm x 4.0 mm i.d.) with column temperature of 40 degrees C; a mixture of 20 mmol/L sodium acetate-methanol-acetonitrile as mobile phase with a flow rate of 1 mL/min and detection at 338 nm. The results indicate that GLPIA2 is rich in acidic amino acid (Glu) and low in aromatic amino acids (Phe and Tyr). The interaction of some proteinases with GLPIA2 was investigated. The inhibitors are more potent against pepsin and yeast proteinase A than other proteinases.