TY - JOUR
T1 - [58] Endo-β-galactosidase from Flavobacterium keratolyticus
AU - Kitamikado, Manabu
AU - Ito, Makoto
AU - Li, Yu Teh
N1 - Funding Information:
Tills work was supported by Grant PCM-79-22466 from the National Science Foundation and Grants NS09626 and RR 00164 from the National Institutes of Health.
Funding Information:
This work was supported by Grant PCM-79-22466f rom the National Science Foundation and Grants NS09626 and RR 00164 from the National Institutes of Health.
PY - 1982/1/1
Y1 - 1982/1/1
N2 - 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.
AB - 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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U2 - 10.1016/0076-6879(82)83060-7
DO - 10.1016/0076-6879(82)83060-7
M3 - Article
C2 - 6808310
AN - SCOPUS:0020023192
SN - 0076-6879
VL - 83
SP - 619
EP - 625
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -