[Purification and characteristic of proteinase inhibitor GLPIA2 from Ganoderma lucidum by submerged fermentation].
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.
Tian
Yaping
Y
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036, China. yapingtian@hotmail.com
Zhang
Kechang
K
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D011480:Protease Inhibitors / Q000032:analysis*; D020940:Reishi / Q000737:chemistry*
D011480:Protease Inhibitors