PURIFICATION FROM FUSOBACTERIUM-MORTIFERUM ATCC-25557 OF A L-O-ALPHA-D-GLUCOPYRANOSYL-6-PHOSPHOGLUCOHYDROLASE THAT HYDROLYZES MALTOSE 6-PHOSPHATE AND RELATED PHOSPHO-ALPHA-D-GLUCOSIDES
J. Thompson et al., PURIFICATION FROM FUSOBACTERIUM-MORTIFERUM ATCC-25557 OF A L-O-ALPHA-D-GLUCOPYRANOSYL-6-PHOSPHOGLUCOHYDROLASE THAT HYDROLYZES MALTOSE 6-PHOSPHATE AND RELATED PHOSPHO-ALPHA-D-GLUCOSIDES, Journal of bacteriology, 177(9), 1995, pp. 2505-2512
l-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (8-phospho-alpha-gl
ucosidase) has been purified from Fusobacterium mortiferum ATCC 25557,
p-Nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alpha Glc6P) s
erved as the chromogenic substrate for detection and assay of enzyme a
ctivity, The O-2-sensitive, metal-dependent phospho-alpha-glucosidase
was stabilized during purification by inclusion of dithiothreitol and
Mn2+ ion in chromatography buffers. Various 6-phosphoryl-O-alpha-linke
d glucosides, including maltose 6-phosphate, pNP alpha Glc6P, trehalos
e 6-phosphate, and sucrose 6-phosphate, mere hydrolyzed by the enzyme
to yield D-glucose 6-phosphate and aglycone moieties in a 1:1 molar ra
tio, 6-Phospho-alpha-glucosidase (M(r) of similar to 49,000; pI of sim
ilar to 4.9) is activated by Fe2+, Mn2+, Co2+, and Ni2+, and the maxim
um rate of pNP alpha Glc6P hydrolysis occurs at 40 degrees C within th
e pH range 7.0 to 7.5, The sequence of the first 32 amino acids of 6-p
hospho-alpha-glucdsidase exhibits 67% identity (90% similarity) to tha
t deduced for the N terminus of a putative phospho-beta-glucosidase (d
esignated ORF f212) encoded by glvG in Escherichia coli. Western blots
involving highly specific polyclonal antibody against 6-phospho-alpha
-glucosidase and spectrophotometric analyses with pNP alpha Glc6P reve
aled only low levels of the enzyme in glucose-, mannose-, or fructose-
grown cells of F. mortiferum, Synthesis of 6-phospho-alpha-glucosidase
increased dramatically during growth of the organism on alpha-glucosi
des, such as maltose, alpha-methylglucoside, trehalose, turanose, and
palatinose.