Separation and purification of trehalase inhibitor from fermentation broth of streptomyces albus

Abstract

Trehalase inhibitors due to their anti-fungal and insecticidal activities are considered to be valuable compounds in the plant protection industry. The major objectives of this study were to isolate a trehalase inhibitor from the broth of Streptomyces albus culture, and to identify its molecular structure. A combined separation process composed of solvent extraction, ion exchange chromatography, ultrafiltration and high performance liquid chromatography (HPLC) has been developed. An improved protocol has been also proposed for trehalase inhibitor assay. Lipids and proteins in micro-filtered broth sample were removed by extraction using butanol and methanol, respectively. The pretreated broth sample was treated by cation exchange chromatography with Dowex 50WX2 resin to recover the inhibitor that was in a protonated form under the given condition, and then by anion exchange chromatography with Amberlite IRA-67 resin to remove impurities. The ion-exchanged sample was filtered with an ultrafiltration membrane (MWCO 500) to remove large molecule impurities. The filtrate was fractionized by HPLC with $NH_2$ Luna column. The fraction with a retention time range of 11.33 - 11.89 min was picked and further analyzed for the identification of the inhibitor. Its molecular weight was found to be 318 - 330, which is different from that of salbostatin that was generally known to be produced by Streptomyces albus. It was observed by NMR and IR analysis to have amino- and sugar-alcohol structure like salbostatin.