[56] Endo-β-N-acetylglucosaminidase L from Streptomyces plicatus

摘要: Publisher Summary This chapter presents the procedure for purification and assay of endo-β-N acetylglucosaminidase L from Streptomyces plicatus. For purification, plicatus is grown at 30 ° on particulate chitin in l-liter batches as described previously Endo-H. Eighteen liters culture media are filtered to yield 15 clear yellow fluid, containing about 1.2 mg protein 11 milliunits Endo-L/ml. The steps involve following: zinc acetate ammonium sulfate precipitations; pH4.2 precipitation; agarose Chromatography; sulfopropyl-Sephadex C-25 chromatography; phenyl-Sepharose CL-4B chromatography. Endo-L obtained a 40% with an overall 100-fold. On electrophoresis nondenaturing Tris-glycine acrylamide gel or SDS slab electrophoresis, single band evident step, however, when combined this step subjected thin-layer isolectric focusing, two species enzyme resolved, one point 4.20, other 4.25. based release [ 3 H]dansyl-AsnGlcNAc substrate H]dansyl-Asn(GlcNAc)2Man. products separated by paper chromatography 1-butanol, ethanol, water located their fluorescence under UV light. H]Dansyl- AsnGlcNAc eluted quantitated liquid scintillation spectrometry.