Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083

Abstract

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards alpha-glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while alpha-glucose-1-phosphate served as donor. D- and L-arabinose, D- and L-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with D-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was alpha-Glcp(1-->1)beta-Araf.

Bifidobacterium longum Requires a Fructokinase (Frk; ATP: d -Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

“…Bioinformatic analyses complement biochemical data reporting the purification and characterization of Bifidobacterium sp. enzymes that are capable of hydrolyzing various glycosidic bonds from plant-derived carbohydrates and are often inducible by the released monomeric molecules (15,22,29,33,41,48,56,58,63). Moreover, several studies conducted on fructose-containing polymers as potential selective substrates for colonic bacteria have provided evidence that bifidobacteria are able to ferment these carbohydrates, in particular the short linear chains of ␤-2-1-linked fructosyl units (7,24,30,36,37).…”

Carbohydrate metabolism in Bifidobacteria

“…Several studies have shown that sucrose phosphorylase from B. adolescentis DSM20083 [144], B. animalis subsp. lactis [132] and B. longum subsp.…”

“…In addition, these characterized sucrose phosphorylases were shown to display transglycosylation activity with glucose-1-phosphate as a donor and monomeric sugars, such as D-and L-arabinose, D-and L-arabitol, and xylitol in the case of B. longum subsp. longum [144], as acceptors.…”

Glycosyl hydrolases from Bifidobacterium adolescentis DSM20083. An overview

“…Depending on the enzyme, hydrolysis often exceeds transglycosylation. Experiments in our laboratory have shown that α-galactosidase from B. adolescentis is a rather efficient transglycosylase in comparison with the other retaining glycosyl hydrolyses [9,[30][31][32]35]. Therefore, α-galactosidase was chosen for further improvement of its transglycosylation activity.…”

“…Therefore, sucrose phosphorylase might play an important role in the fermentation pathway of sucrose obtained after hydrolysis of raffinose and stachyose. Sucrose phosphorylases genes have been cloned from B. adolescentis [32], B. lactis [29], and B. longum [10].…”