A novel alpha-galactosidase from Bifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression.
A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of approximately 243 kDa and a subunit size of approximately 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (approximately 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) > or = 3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).
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
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001644:Bifidobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016415:Sequence Alignment; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D000519:alpha-Galactosidase