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Enzymatic synthesis and characterization of fructooligosaccharides and novel maltosylfructosides by inulosucrase from Lactobacillus gasseri DSM 20604.

ABSTRACT: The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucrose and sucrose-maltose mixtures was investigated after optimization of synthesis conditions, including enzyme concentration, temperature, pH, and reaction time. The maximum formation of FOS, which consist of ?-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained after 24 h of reaction at 55°C in the presence of sucrose (300 g liter(-1)) and 1.6 U ml(-1) of IS-25 mM sodium acetate buffer-1 mM CaCl2 (pH 5.2). The production of MFOS was also studied as a function of the initial ratios of sucrose to maltose (10:50, 20:40, 30:30, and 40:20, expressed in g 100 ml(-1)). The highest yield in total MFOS was attained after 24 to 32 h of reaction time and ranged from 13% (10:50 sucrose/maltose) to 52% (30:30 sucrose/maltose) in weight with respect to the initial amount of maltose. Nuclear magnetic resonance (NMR) structural characterization indicated that IS from L. gasseri specifically transferred fructose moieties of sucrose to either C-1 of the reducing end or C-6 of the nonreducing end of maltose. Thus, the trisaccharide erlose [?-d-glucopyranosyl-(1?4)-?-d-glucopyranosyl-(1?2)-?-d-fructofuranoside] was the main synthesized MFOS followed by neo-erlose [?-d-fructofuranosyl-(2?6)-?-d-glucopyranosyl-(1?4)-?-d-glucopyranose]. The formation of MFOS with a higher degree of polymerization was also demonstrated by the transfer of additional fructose residues to C-1 of either the ?-2,1-linked fructose or the ?-2,6-linked fructose to maltose, revealing the capacity of MFOS to serve as acceptors.

SUBMITTER: Diez-Municio M

PROVIDER: S-EPMC3697562 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Enzymatic synthesis and characterization of fructooligosaccharides and novel maltosylfructosides by inulosucrase from Lactobacillus gasseri DSM 20604.

Díez-Municio Marina M de las Rivas Blanca B Jimeno Maria Luisa ML Muñoz Rosario R Moreno F Javier FJ Herrero Miguel M

Applied and environmental microbiology 20130503 13

The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucrose and sucrose-maltose mixtures was investigated after optimization of synthesis conditions, including enzyme concentration, temperature, pH, and reaction time. The maximum formation of FOS, which consist of β-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained afte ...[more]

PMID: 23645191

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Project description:Lactobacillus gasseri LA327, isolated from the large intestine tissue in humans, is a bacteriocinogenic strain with two kinds of class IIb bacteriocin structural genes, i.e., those for gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A from L. gasseri LA327 revealed that the amino acid sequences for GT corresponded with those for GT genes, except for GatK (histidine kinase). However, Acd LF221A genes had analogues which differed in at least one amino acid residue, to encode a class IIb bacteriocin designated gassericin S (GS). The LA327 strain retained antimicrobial activity after the deletion of the GT structural genes (gatAX); however, both GS and GT activities were lost by deletion of the putative ABC transporter gene (gatT). This indicates that the LA327 strain produces GS and GT and that GS secretion is performed via GT genes with the inclusion of gatT Homologous expression using deletion mutants of GS and GT, each containing a single peptide, elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins and that no synergistic activity was observed between GS and GT peptides. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, the stability of expressed GS to pH, heat, and protease was determined.IMPORTANCE Bacteriocins are regarded as potential alternatives for antibiotics in the absence of highly resistant bacteria. In particular, two-peptide (class IIb) bacteriocins exhibit the maximum activity through the synergy of two components, and their antimicrobial spectra are known to be relatively wide. However, there are few reports of synergistic activity of class IIb bacteriocins determined by isolation and purification of individual peptides. Our results clarified the interaction of each class IIb component peptide for GT and GS via the construction of homologous mutants, which were not dependent on the purification. These data may contribute to understanding the mechanisms of action by which class IIb bacteriocins exhibit wide antibacterial spectra.

Dataset Information

Enzymatic synthesis and characterization of fructooligosaccharides and novel maltosylfructosides by inulosucrase from Lactobacillus gasseri DSM 20604.

Publications

Enzymatic synthesis and characterization of fructooligosaccharides and novel maltosylfructosides by inulosucrase from Lactobacillus gasseri DSM 20604.

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