This chapter presents the procedure for purification and assay of endo-β-galactosidase from Flavobacterium keratolyticus. The organism from the slant culture is used to inoculate 50-ml flasks each containing l0 ml of liquid medium. The flasks are incubated without shaking at 25 ° for 2 days. Each culture is then transferred to a 2-liter flask containing 1 liter of liquid medium, and incubated stationary at the same temperature for 5 days. All flasks are plugged with cotton. The cultures are centrifuged at 17,000g for 30 rain. From 15 liters of the liquid culture, 14,060 ml of a clear culture supernatant are obtained. The steps of purification are (1) ammonium sulfate precipitation, (2) sephadex G-100 chromatography, (3) chromatography on a combined column of CM-Sephadex C-50 and DEAE-Sephadex A-50, (4) matrex gel Blue A chromatography, and (5) DEAE-Sephadex A-50 chromatography. The final preparation shows one major band on polyacrylamide gel electrophoresis. Keratan sulfate isolated from whale nasal cartilage is used as substrate in assay.
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