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Two ?-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

ABSTRACT: Two ?-galactosidases, ?-gal I and ?-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both ?-gal I and ?-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-?-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for ?-gal I, and at pH 6.5 and 55°C for ?-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for ?-gal I, and 543 and 25 mM-1s-1 for ?-gal II. Both ?-gal I and ?-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with ?-gal I and ?-gal II, respectively. The predominant transgalactosylation products are ?-D-Galp-(1?6)-D-Glc (allolactose) and ?-D-Galp-(1?3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by ?-gal I and ?-gal II, respectively, indicating that both enzymes have a propensity to synthesize ?-(1?6) and ?-(1?3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

SUBMITTER: Arreola SL

PROVIDER: S-EPMC4121272 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

Arreola Sheryl Lozel SL Intanon Montira M Suljic Jasmina J Kittl Roman R Pham Ngoc Hung NH Kosma Paul P Haltrich Dietmar D Nguyen Thu-Ha TH

PloS one 20140804 8

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two ...[more]

PMID: 25089712

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Project description:Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of beta-GalI and beta-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).

Dataset Information

Two ?-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

Publications

Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

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