Purification and partial characterization of alpha-L-arabinofuranosidase produced by Thermomonospora fusca.
Thermomonospora fusca produced a relatively high level of alpha-L-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparent Km value for the crude alpha-L-arabinofuranosidase preparation was 180 mumol/L 4-nitrophenyl alpha-L-arabinofuranoside; the upsilon lim value was the release of 40 mumol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of approximately 92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular alpha-L-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified alpha-L-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.
Tuncer
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M
Biyoloji Bölümü, Fen-Edebiyat Fakültesi, Mersin Universitesi, 33342 Mersin, Turkey. mtuncer@mersin.edu.tr
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D000192:Actinomycetales / Q000201:enzymology* / Q000254:growth & development; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007525:Isoelectric Focusing; D014990:Xylans / Q000378:metabolism
D014990:Xylans; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase