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Title: Influence of human gut microbiota on the metabolic fate of glucosinolates
Author: Luang-In, Vijitra
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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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.
Supervisor: Rossiter, John ; Buck, Martin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available