The Purification of Grifolan Synthase and the Determination of its Molecular Weight

Abstract

Purpose To search for a purification method of Grifolan Synthase crude extract from the mycelium of Grifola frondosa and determine its molecular weight. Methods Grifolan Synthase crude extract from the mycelium of Grifola frondosa was purified by recrystallization and native-PAGE. The process is as follows: Grifolan Synthase crude extract was dissolved in buffer solution (pH7.0). 10ml Grifolan Synthase crude extract solution was mixed with ammonium sulfate slowly to the concentration of 60%, and then mixed with 3ml cooling acetone (refrigeration at -18 °C for 12 hours) slowly, after standing for 24 hours, centrifugated at 5000rpm for 10 minutes at 4°C. Material at the interface was collected and air-dried as higher purity Grifolan Synthase. The higher purity Grifolan Synthase was dispersed in the native-PAGE gel, and the active band of native-PAGE gel was cut down and broken by ultrasonic treatment for 1min, then centrifugated at 5000rpm for 10 minutes at 4°C, the supernatant was taken and mixed with ammonium sulfate slowly to the concentration of 60%, after 24 hours on standing, centrifugated at 12000rpm for 10 minutes at 4°C, the pellets was got and the purity was checked by SDS-PAGE. The molecular weight of Grifolan Synthase was determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Results Grifolan Synthase purified by recrystallization and native-PAGE was checked to be a single band by SDS-PAGE. The data of molecular weight obtained by SDS-PAGE showed that the molecular weight of Grifolan Synthase was 55000Da. Conclusion A purification method of Grifolan Synthase crude extract from the mycelium of Grifola frondosa was researched out, and the molecular weight of grifolan synthase was studied in this paper. It can lay the foundation for the further study on the structure and function of Grifolan Synthase.