Purification and partial characterization of α-L-arabinofuranosidase produced by Thermomonospora fusca

Abstract

Thermomonospora fusca produced a relatively high level of α-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 α-L-arabinofuranosidase preparation was 180 μmol/L 4-nitrophenyl α-L-arabinofuranoside; the υlim value was the release of 40 μmol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of ≈92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular α-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 α-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%.