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    Identification of Proteins Possibly Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8
    (2015) Luang-In, Vijitra; Narbad, Arjan; Cebeci, Fatma; Bennett, Mark; Rossiter, John T.
    This study was aimed to identify sinigrin-induced bacterial proteins potentially involved in the metabolism of glucosinolate in two glucosinolate-metabolising bacteria Lactobacillus agilis R16 and Escherichia coli VL8. Sinigrin (2 mM) was used to induce the proteins in both bacteria under anaerobic incubation for 8 h at 30 C for L. agilis R16 and 37 C for E. coli VL8 and the controls without sinigrin were performed. Allyl isothiocyanate and allyl nitrile as two degradation products of sinigrin were detected in sinigrin-induced cultures of L. agilis R16 (27 % total products) and E. coli VL8 (38 % total products) from a complete sinigrin degradation in 8 h for both bacteria. 2D gel electrophoresis was conducted to identify induced proteins with at least twofold increased abundance. Sinigrin-induced L. agilis R16 and the control produced 1561 and 1543 protein spots, respectively. For E. coli VL8, 1363 spots were detected in sinigrin-induced and 1354 spots in the control. A combination of distinct proteins and upregulated proteins of 32 and 35 spots in L. agilis R16 and E. coli VL8, respectively were detected upon sinigrin induction. Of these, 12 and 16 spots from each bacterium respectively were identified by LC–MS/MS. In both bacteria most of the identified proteins are involved in carbohydrate metabolism, oxidoreduction system and sugar transport while the minority belong to purine metabolism, hydrolysis, and proteolysis. This indicated that sinigrin induction led to the expressions of proteins with similar functions in both bacteria and these proteins may play a role in bacterial glucosinolate metabolism.
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    Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1
    (Springer, 2022) Cebeci, Fatma; Mayer, Melinda J.; Rossiter, John T.; Mithen, Richard; Narbad, Arjan
    Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 degrees C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. K-m and V-max were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.
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    Utilisation of an active branching sucrase from Lactobacillus kunkeei AP-37 to produce techno-functional poly-oligosaccharides
    (Elsevier, 2023) Ispirli, Huemeyra; Korkmaz, Kader; Arioglu-Tuncil, Seda; Bozkurt, Fatih; Sagdic, Osman; Tuncil, Yunus Emre; Narbad, Arjan
    Glucansucrase AP-37 was extracted from the culture supernatant of Lactobacillus kunkeei AP-37 and character-istics of the glucan produced by the active glucansucrase in terms of structural and functional roles were determined in this study. A molecular weight around 300 kDa was observed for glucansucrase AP-37 and its acceptor reactions with maltose, melibiose and mannose were also conducted to unveil the prebiotic potential of the poly-oligosaccharides formed via these reactions. The core structure of glucan AP-37 was determined by 1H and 13C NMR and GC/MS analysis which revealed that glucan AP-37 was a highly branched dextran composing of high levels of (1-+ 3)-linked alpha-D-glucose units with low levels of (1-+ 2)-linked alpha-D-glucose units. The structural features of the glucan formed, demonstrated that glucansucrase AP-37 was an alpha-(1-+ 3) branching sucrase. Dextran AP-37 was further characterised by FTIR analysis and XRD analysis demonstrated its amorphous nature. A fibrous compact morphology was observed for dextran AP-37 with SEM analysis whereas TGA and DSC analysis revealed its high stability as no degradation was observed up to 312 degrees C. Finally, the prebiotic potential of the dextran AP-37 and the gluco-oligosaccharides produced with the acceptor reaction of alpha-(1-+ 3) branching sucrase AP-37 were determined and promising results were found for the gluco-oligosaccharides to act as prebiotics.