Andrew P. Laws

The relevance of exopolysaccharides to the rheological properties in milk fermented with ropy strains of lactic acid bacteria

The subunit structures from a number of different species of lactic acid bacteria are now known, with many of the structures being published only in the last five years. The nuclear magnetic resonance spectra to date show around twenty different subunits and molecular mass determinations suggest that these exopolysaccharides occur as very large molecules, frequently greater than 1×106 Da. The physical properties of these molecules will be influenced by their mass and also by the subunit structure. Milk however is a complex medium and studies concerned with effects on rheology of milk as a result of in situ production of exopolysaccharides from these bacteria have to consider many factors. Studies have found that it is not only the nature of the exopolysaccharide, but that the amount produced, the acidity of the milk, the composition of the milk and the length of fermentation, also influence final texture.

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure.

Aims: To compare galactose‐negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs).

Structural characterisation of the exopolysaccharide produced by Streptococcus thermophilus EU20

Streptococcus thermophilus EU20 when grown on skimmed milk secretes a high-molecular-weight exopolysaccharide that is composed of glucose, galactose and rhamnose in a molar ratio of 2:3:2. Using chemical techniques and 1D and 2D-NMR spectroscopy (1H and 13C) the polysaccharide has been shown to possess a heptasaccharide repeating unit having the following structure: [chemical structure: see text]. Treatment of the polysaccharide with mild acid (0.5 M TFA, 100 degrees C for 1 h) liberates two oligosaccharides; the components correspond to the repeating unit and a hexasaccharide equivalent to the repeating unit minus the terminal alpha-L-Rhap.

Cysteinyl peptide Inhibitors of Bacillus cereus Zinc β-Lactamase

Several cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus metallo-β-lactamase. The pH dependence of pKi indicates that the thiol anion displaces hydroxide ion from the active site zinc(II). , -Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the active site

The mechanism of catalysis and the inhibition of β-lactamases

Formation of a tetrahedral intermediate by nucleophilic attack on the β-lactam carbonyl carbon of penicillins generates a lone pair on the β-lactam nitrogen which is syn to the incoming nucleophile, in contrast to the normal anti arrangement found in peptides. Ring opening of the β-lactam requires protonation of the β-lactam nitrogen by a general acid catalyst. The general acid/base catalyst in β-lactamases is probably a glutamate and a tyrosine residue in class A and C enzymes, respectively. Phosphonamidates inactivate class C β-lactamases by phosphonylation of the active site serine, the rate of which is enhanced by a factor of at least 106. The enzyme’s catalytic machinery used for hydrolysis is also used for phosphonylation. The rate enhancement may be greater than 109 if the mechanism occurs by an inhibitor assisted reaction involving intramolecular general acid catalysis. Class B metallo-β-lactamases are inhibited by thiol derivatives with Ki as low as 10 µM. The mechanism of hydrolysis of the metallo-β-lactamase involves a dianionic tetrahedral intermediate stabilised by zinc(II).

The Chemical Reactivity of β-Lactams, β-Sultams and β-Phospholactams

β-Lactam antibiotics display a range of biological activities. The origin of this diverse biological activity is discussed with reference to the chemical reactivity of the small ring system. The reactions of β-sultams and β-phospholactams with simple nucleophiles are reported and their potential as mechanism based inhibitors of bacterial and mammalian serine proteases is described.

Structure of the high molecular weight exopolysaccharide produced by Bifidobacterium animalis subsp. lactis IPLA-R1 and sequence analysis of its putative eps cluster

The bile adapted strain Bifidobacterium animalis subsp. lactis IPLA-R1 secretes a high molecular weight exopolysaccharide (HMW-EPS) when grown on the surface of agar-MRSC. This EPS is composed of L-rhamnopyranosyl, D-glucopyranosyl, D-galactopyranosyl and D-galactofuranosyl residues in the ratio of 3:1:1:1. Linkage analysis and 1D and 2D NMR spectroscopy were used to show that the EPS has a hexasaccharide repeating unit with the following structure: [See formula in text]. Treatment of the EPS with mild acid cleanly removed the terminal d-galactofuranosyl residue. The eps cluster sequenced for strain IPLA-R1 showed high genetic homology with putative eps clusters annotated in the genomes of strains from the same species. It is of note that several genes coding for rhamnose-precursors are present in the eps cluster, which could be correlated with the high percentage of rhamnose detected in its EPS repeated unit.

UDP-N-Acetylglucosamine 4-Epimerase Activity Indicates the Presence of N-Acetylgalactosamine in Exopolysaccharides of Streptococcus thermophilus Strains

ABSTRACT The monomer composition of the exopolysaccharides (EPS) produced byStreptococcus thermophilus LY03 and S. thermophilus Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and N-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-N-acetylglucosamine 4-epimerase, converting UDP-N-acetylglucosamine into UDP-N-acetylgalactosamine, is responsible for the presence of N-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-N-acetylglucosamine 4-epimerase was higher in bothS. thermophilus strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during S. thermophilus Sfi20 fermentations. A correlation between activities of the enzymes α-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for S. thermophilus LY03, although S. thermophilusSfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain.

Lactobacillus-produced exopolysaccharides and their potential health benefits: a review.

Lactic acid bacteria, such as those of the Lactobacillus genus, naturally reside within the microbiota of the human body and have long been used as starter cultures and probiotic enhancers in fermented foods, such as fermented drinks, yoghurts and cheeses. Many of the beneficial qualities of these bacteria have traditionally been associated with the bacteria themselves, however, a recent spate of studies have demonstrated a wide variety of biological effects exhibited by lactobacilli-produced exopolysaccharides which could, theoretically, confer a range of local and systemic health benefits upon the host. In this review, we discuss the production of exopolysaccharides within the Lactobacillus genus and explore their potential as beneficial bioactive compounds.

Structural characterisation of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterisation of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23

The exopolysaccharide (EPS) from Lactobacillus delbrueckii subsp. bulgaricus EU23 was perdeuteriomethylated and the perdeuteriomethylated EPS (pdm-EPS) purified by elution from a C(18) Sep-Pak cartridge. Both 1D and 2D NMR spectra were recorded for the pdm-EPS and these were interpreted to provide assignments for the individual 1H and 13C resonances of the sugar residues of the repeating unit. Using a combination of the results from monomer analysis and linkage analysis of the native EPS and the ROESY and HMBC NMR spectra of the pdm-EPS the following structure has been determined for the repeating unit:A process for characterising polysaccharides having low solubility in aqueous solution is reported.