Purification and characterization of an alpha-L-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA).
The gene encoding an alpha-L-arabinofuranosidase (abfA) was homologously cloned in Streptomyces lividans and its DNA sequence was determined. The enzyme was purified from the cytoplasm of the hyperproducing clone S. lividans IAF116. Its M(r) was estimated by gel filtration and found to be approx. 380,000. Since SDS/PAGE indicated a native protein of M(r) 69,000, it can be concluded that the native protein consists of several subunits of that size. The pI value was 4.6. The kinetic constants determined with p-nitrophenyl alpha-L-arabinofuranoside as substrate were a Vmax of 180 units/mg of protein and a Km of 0.6 mM. The specific activity of the purified enzyme on this substrate was 153 units/mg of protein. Optimal enzyme activity was obtained at 60 degrees C and pH 6.0. The enzyme cleaved p-nitrophenyl alpha-L-arabinofuranoside, but had no activity on a variety of other p-nitrophenyl glycosides, except on p-nitrophenyl beta-D-xylopyranoside. The enzyme showed no activity on oat-spelts (Avena sativa) xylan or arabinogalactan, but acted on beet (Beta) arabinan or arabinoxylan. Hydrolysis occurred on arabino-oligoxylosides obtained from oat-splets xylan after digestion with xylanases. Since S. lividans normally does not secrete arabinofuranosidase, this enzyme may play a role in the assimilation of arabinose moieties from arabinose-containing xylo-oligosaccharides generated by beta-xylosidases or xylanases.
Manin
C
C
Centre de recherche en microbiologie appliquée, Institut Armand-Frappier, Université du Québec, Laval-des-Rapides.
Shareek
F
F
Morosoli
R
R
Kluepfel
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016622:Silver Staining; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D003470:Culture Media; D018076:DNA, Complementary; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase