Expression and characterization of a Bifidobacterium adolescentis beta-mannanase carrying mannan-binding and cell association motifs.
The gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k(cat) was determined to be 444 s(-1) and K(m) 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.
Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, Lund, Sweden.
Evelina
E
Kulcinskaja
Anna
A
Rosengren
Romany
R
Ibrahim
Katarína
K
Kolenová
Henrik
H
Stålbrand
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D002415:Cats; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008351:Mannans / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D044902:beta-Mannosidase / Q000235:genetics* / Q000378:metabolism*
D008351:Mannans; D011994:Recombinant Proteins; C012990:galactomannan; D044902:beta-Mannosidase; D008358:Mannose; D005690:Galactose