Comparative analysis of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation

Goulas, T., Goulas, A., Tzortzis, G. and Gibson, G. R. ORCID: https://orcid.org/0000-0002-0566-0476 (2009) Comparative analysis of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation. Applied Microbiology and Biotechnology, 82 (6). pp. 1079-1088. ISSN 0175-7598 doi: 10.1007/s00253-008-1795-5

Abstract/Summary

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.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/13354
Item Type	Article
Refereed	Yes
Divisions	Life Sciences > School of Chemistry, Food and Pharmacy > Department of Food and Nutritional Sciences
Uncontrolled Keywords	Bifidobacterium bifidum, Bifidobacteria, Galactosidase, Prebiotic, MOLECULAR-CLONING, GALACTOOLIGOSACCHARIDES, INFANTIS, LACTOSE
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