Influence of human gut microbiota on the metabolic fate of glucosinolates
File(s)
Author(s)
Luang-In, Vijitra
Type
Thesis
Abstract
Glucosinolates (GSLs) are secondary metabolites predominantly found in cruciferous vegetables such
as broccoli, brussel sprout, cabbage and cauliflower which upon chopping and chewing will release
the indigenous plant myrosinase enzyme that catalyzes the hydrolysis of GSLs. This hydrolysis
releases a range of breakdown products including isothiocyanates (ITCs), which have been implicated
in the cancer-protective effects of cruciferous vegetables. Certain human gut bacteria are able to
metabolize GSLs and produce ITCs for human health benefits. In this work, six GSL-metabolizing
bacterial strains were isolated from human faecal sample and identified. Most bacteria were capable
of producing both nitriles (NITs) and ITCs from the metabolism of GSLs however Enterococcus sp.
C213 and Enterococcus faecium KT4S13 produced only NITs. Enterococcus casseliflavus NCCP-53,
Escherichia coli O83:H1 NRG 857C and Lactobacillus agilis R16 were able to metabolize different
types (allyl, aromatic, methylthioalkyl, methylsulfinylalkyl and indolyl) of GSLs differently over
24 h of in vitro anaerobic fermentations. For all GSLs, ITC production seemed to peak between 4 and
8 h of incubation and then declined due to the inherent instability of ITCs in culture broths and
buffers. In contrast, NIT productions gradually increased over time and remained relatively
constant. The total percentage products from each GSL metabolism in all three bacteria never
reached 100%. Interestingly, E. coli O83:H1 NRG 857C produced methylthioalkyl ITCs and NITs from
methylsulfinylalkyl GSLs while E. casseliflavus NCCP-53 produced only methylsulfinylalkyl ITCs from
the same GSLs. This difference was due to reductase activity in E. coli O83:H1 NRG 857C intact
cells and cell-free extracts that biotransforms the sulfoxide groups of methylsulfinylalkyl GSLs to
the sulfide groups. The reductase enzyme is yet to be identified at the gene and protein level,
however it has been characterized using
cell-free extracts in this work. This reductase is inducible by GSLs, oxygen-independent and
requires Mg2+ ion and NADP(H) as co-factors for its activity with optimum pH and temperature at pH
7.0 and 37˚C, respectively. Arylsulfatase activity was also detected in this bacterium. The
corresponding recombinant SUL2 enzyme (57 kDa) of E. coli O83:H1 NRG 857C expressed in BL21(DE3)
exhibited arylsulfatase activity by desulfating synthetic p-nitrocatachol sulfate substrate with
optimum pH and temperature at pH 6.0 and 30˚C,
respectively. In addition, GSL-sulfatase activity was detected in crude extracts by being able to desulfate different intact GSLs to produce desulfo-glucosinolates (DS-GSLs) with less
efficiency in comparison with the commercially available snail sulfatase from Helix pomatia. The
catalytic efficiency of recombinant SUL2 enzyme for GSLs in descending order is as follows;
sinigrin > glucoerucin > gluconasturtiin > glucoiberin. The DS-GSLs (except DS-glucoraphanin) then
act as substrates for the recombinant GH3 enzyme defived from E. casseliflavus NCCP-53 to produce
the corresponding NIT products in NB broths and the buffer with the presence of 5 mM Fe2+ ions.
This enzyme (79 kDa) showed β-O-glucosidase activity for p-nitrophenyl β-D-glucopyranoside with
optimum pH and temperature at pH 7.0 and 37˚C, respectively. NIT productions only occurred from the
metabolism of intact GSLs in bacterial culture broths, but not in the buffers unless 5 mM Fe2+ ions
are present as co-factors. Putative bacterial GSL-degrading enzymes responsible for ITC and NIT
productions from GSL metabolisms are inducible by GSL in resting cells experiments. By using
two-dimensional gel electrophoresis (2-DE) and liquid chromatography mass spectrometry (LC-MS/MS)
for the comparative analysis between proteins obtained from cultures of L. agilis R16 and E. coli
O83:H1 NRG 857C with and without GSL supplementation, upregulated/distinct proteins that may be involved in the metabolism of GSLs by these bacteria were identified. These proteins belong to
(sugar) transport system, carbohydrate metabolism especially kinases and oxidoreduction process. To date, bacterial GSL-degrading enzyme is yet to be identified.
as broccoli, brussel sprout, cabbage and cauliflower which upon chopping and chewing will release
the indigenous plant myrosinase enzyme that catalyzes the hydrolysis of GSLs. This hydrolysis
releases a range of breakdown products including isothiocyanates (ITCs), which have been implicated
in the cancer-protective effects of cruciferous vegetables. Certain human gut bacteria are able to
metabolize GSLs and produce ITCs for human health benefits. In this work, six GSL-metabolizing
bacterial strains were isolated from human faecal sample and identified. Most bacteria were capable
of producing both nitriles (NITs) and ITCs from the metabolism of GSLs however Enterococcus sp.
C213 and Enterococcus faecium KT4S13 produced only NITs. Enterococcus casseliflavus NCCP-53,
Escherichia coli O83:H1 NRG 857C and Lactobacillus agilis R16 were able to metabolize different
types (allyl, aromatic, methylthioalkyl, methylsulfinylalkyl and indolyl) of GSLs differently over
24 h of in vitro anaerobic fermentations. For all GSLs, ITC production seemed to peak between 4 and
8 h of incubation and then declined due to the inherent instability of ITCs in culture broths and
buffers. In contrast, NIT productions gradually increased over time and remained relatively
constant. The total percentage products from each GSL metabolism in all three bacteria never
reached 100%. Interestingly, E. coli O83:H1 NRG 857C produced methylthioalkyl ITCs and NITs from
methylsulfinylalkyl GSLs while E. casseliflavus NCCP-53 produced only methylsulfinylalkyl ITCs from
the same GSLs. This difference was due to reductase activity in E. coli O83:H1 NRG 857C intact
cells and cell-free extracts that biotransforms the sulfoxide groups of methylsulfinylalkyl GSLs to
the sulfide groups. The reductase enzyme is yet to be identified at the gene and protein level,
however it has been characterized using
cell-free extracts in this work. This reductase is inducible by GSLs, oxygen-independent and
requires Mg2+ ion and NADP(H) as co-factors for its activity with optimum pH and temperature at pH
7.0 and 37˚C, respectively. Arylsulfatase activity was also detected in this bacterium. The
corresponding recombinant SUL2 enzyme (57 kDa) of E. coli O83:H1 NRG 857C expressed in BL21(DE3)
exhibited arylsulfatase activity by desulfating synthetic p-nitrocatachol sulfate substrate with
optimum pH and temperature at pH 6.0 and 30˚C,
respectively. In addition, GSL-sulfatase activity was detected in crude extracts by being able to desulfate different intact GSLs to produce desulfo-glucosinolates (DS-GSLs) with less
efficiency in comparison with the commercially available snail sulfatase from Helix pomatia. The
catalytic efficiency of recombinant SUL2 enzyme for GSLs in descending order is as follows;
sinigrin > glucoerucin > gluconasturtiin > glucoiberin. The DS-GSLs (except DS-glucoraphanin) then
act as substrates for the recombinant GH3 enzyme defived from E. casseliflavus NCCP-53 to produce
the corresponding NIT products in NB broths and the buffer with the presence of 5 mM Fe2+ ions.
This enzyme (79 kDa) showed β-O-glucosidase activity for p-nitrophenyl β-D-glucopyranoside with
optimum pH and temperature at pH 7.0 and 37˚C, respectively. NIT productions only occurred from the
metabolism of intact GSLs in bacterial culture broths, but not in the buffers unless 5 mM Fe2+ ions
are present as co-factors. Putative bacterial GSL-degrading enzymes responsible for ITC and NIT
productions from GSL metabolisms are inducible by GSL in resting cells experiments. By using
two-dimensional gel electrophoresis (2-DE) and liquid chromatography mass spectrometry (LC-MS/MS)
for the comparative analysis between proteins obtained from cultures of L. agilis R16 and E. coli
O83:H1 NRG 857C with and without GSL supplementation, upregulated/distinct proteins that may be involved in the metabolism of GSLs by these bacteria were identified. These proteins belong to
(sugar) transport system, carbohydrate metabolism especially kinases and oxidoreduction process. To date, bacterial GSL-degrading enzyme is yet to be identified.
Version
Open Access
Date Issued
2013-03
Date Awarded
2013-07
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Rossiter, John
Buck, Martin
Creator
Luang-In, Vijitra
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)