Functional screening of human faecal microbiome metagenome library for glycoside hydrolase enzymes

Members of the human gut microbiota use glycoside hydrolase (GH) enzymes, such as sialidases, α-fucosidases and β-galactosidases to forage on host mucin glycans and dietary fibers. A human faecal metagenomic library was constructed and screened to identify new glycoside hydrolase enzymes particularly α-fucosidases, sialidases and β-galactosidases. 33,000 clones were screened for α-fucosidases on LB agar supplemented with X-Fucose as substrate and 11,000 clones were screened on MacConkey base agar supplementd with 2’-Fucosyllactose, however α-fucosidase expressing clones were not detected. Similarly out of 16,128 clones screened for sialidase on MacConkey base agar supplemented with 3’-Sialyllactose and 6’-Sialyllactose, sialidase expressing clones were not detected. However, out of the 16,000 clones screened for β-galactosidases on MacConkey lactose agar, 30 β-galactosidase positive clones were identified. The β-galactosidase gene found in the majority of the clones was BAD_1582, from Bifidobacterium adolescentis. This gene was cloned with the incorporation of a hexa histidine tag and expressed in Escherichia coli. The His-tagged β-galactosidase enzyme was purified using Ni2+-NTA affinity chromatography and size filtration. The enzyme had optimal activity at pH 7.0 and 37°C, with a wide range of pH (4-10) and temperature (0-40oC) stability. The enzyme requires a divalent metal ion cofactor, with the highest activity detected in the presence of Mg2+ and activity was inhibited by the presence of Cu2+ and Mn2+. The kinetic parameters were determined using ortho-nitrophenyl-β-D-galactopyranoside (oNPG) as a substrate. The enzyme had a Vmax of 51 μmol/min/mg and a Km of 2.5 mM. The enzyme was competitively inhibited by lactose with a Ki of 3.0 mM and exhibited low product inhibition by galactose with a Ki of 115 mM and a high tolerance for glucose (66% of its activity retained in the presence of 700 mM of glucose). In addition, this enzyme displayed transgalactosylation activity to produce galactooligosaccharides (GOS) using lactose as a substrate. BAD_1582 was immobilized in calcium alginate and gelatin blend crosslinked with glutaraldehyde. The immobilized BAD_1582 displayed enhanced pH and temperature stability compared to the free enzyme. The aforementioned properties of BAD_1582 makes it an ideal candidate to produce lactose free dairy products and manufacture of GOS.

Funder

College of Science, National University of Ireland, Galway

Publisher

NUI Galway

Rights

Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

Collections

University of Galway Theses (PhD Theses)