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Applied Microbiology and Biotechnology

, Volume 82, Issue 6, pp 1079–1088 | Cite as

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

  • Theodoros Goulas
  • Athanasios Goulas
  • George Tzortzis
  • Glenn R. Gibson
Biotechnologically Relevant Enzymes and Proteins

Abstract

Four different β-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 × 104 s−1 M−1), BbgIII (2.36 × 104 s−1 M−1) and especially BbgIV (4.01 × 105 s−1 M−1) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 104 s−1 M−1) than lactose (1.48 × 103 s−1 M−1). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.

Keywords

Bifidobacterium bifidum Bifidobacteria Galactosidase Prebiotic 

Notes

Acknowledgements

This work was supported by the Greek State Scholarship’s Foundation (IKY).

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Theodoros Goulas
    • 1
  • Athanasios Goulas
    • 1
  • George Tzortzis
    • 1
  • Glenn R. Gibson
    • 1
  1. 1.Department of Food Biosciences, School of Chemistry, Food Biosciences and PharmacyThe University of ReadingReadingUK

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