Comparative analysis of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation.
Four different beta-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4-5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4-6.8). Na(+) and/or K(+) ions were prerequisite for BbgI and BbgIV activity in Bis-Tris-buffered solutions, whereas Mg(++) was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg(++), Mn(++) and Ca(++) ions exerting the most positive effect. Determination of the specificity constants (k(cat)/K(m)) clearly indicated that BbgI (6.11 x 10(4) s(-1) M(-1)), BbgIII (2.36 x 10(4) s(-1) M(-1)) and especially BbgIV (4.01 x 10(5) s(-1) M(-1)) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for beta-D-(1-->6) galactobiose (5.59 x 10(4) s(-1) M(-1)) than lactose (1.48 x 10(3) s(-1) M(-1)). Activity measurements towards other substrates (e.g. beta-D-(1-->6) galactobiose, beta-D-(1-->4) galactobiose, beta-D-(1-->4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the beta-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.
Goulas
Theodoros
T
Department of Food Biosciences, School of Chemistry, Food Biosciences and Pharmacy, The University of Reading, P.O. Box 226, Whiteknights, Reading RG6 6AP, UK. t.goulas@reading.ac.uk
Goulas
Athanasios
A
Tzortzis
George
G
Gibson
Glenn R
GR
D000606:Amino Sugars / Q000378:metabolism; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D002412:Cations / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004187:Disaccharides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D055503:Protein Multimerization; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism*
D000606:Amino Sugars; D002412:Cations; D002413:Cations, Divalent; D003067:Coenzymes; D004187:Disaccharides; D004791:Enzyme Inhibitors; D008670:Metals; C000458:N-acetyllactosamine; C056853:6-O-galactopyranosylgalactose; D001616:beta-Galactosidase; D007785:Lactose