The Lreu_1276 protein from Limosilactobacillus reuteri represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria.

Tetrahydrofolate is a cofactor involved in C1 metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate. Although two structurally distinct DHNTP pyrophosphohydrolases have been identified in the intestinal bacteria Lactococcus lactis and Escherichia coli, the distribution of their homologs is limited. Here, we aimed to identify a third DHNTP pyrophosphohydrolase gene in the intestinal lactic acid bacterium Limosilactobacillus reuteri. In a gene operon including genes involved in dihydrofolate biosynthesis, we focused on the lreu_1276 gene, annotated as Ham1 family protein or XTP/dITP diphosphohydrolase, as a candidate encoding DHNTP pyrophosphohydrolase. The Lreu_1276 recombinant protein was prepared using E. coli and purified. Biochemical analyses of the reaction product revealed that the Lreu_1276 protein displays significant pyrophosphohydrolase activity toward DHNTP. The optimal reaction temperature and pH were 35°C and around 7, respectively. Substrate specificity was relatively strict among 17 tested compounds. Although previously characterized DHNTP pyrophosphohydrolases prefer Mg2+, the Lreu_1276 protein exhibited maximum activity in the presence of Mn2+, with a specific activity of 28.2 ± 2.0 µmol min-1 mg-1 in the presence of 1 mM Mn2+. The three DHNTP pyrophosphohydrolases do not share structural similarity to one another, and the distribution of their homologs does not overlap, implying that the Lreu_1276 protein represents a third structurally novel DHNTP pyrophosphohydrolase in bacteria. IMPORTANCE: The identification of a structurally novel DHNTP pyrophosphohydrolase in L. reuteri provides valuable information in understanding tetrahydrofolate biosynthesis in bacteria that possess lreu_1276 homologs. Interestingly, however, even with the identification of a third family of DHNTP pyrophosphohydrolases, there are still a number of bacteria that do not harbor homologs for any of the three genes while possessing other genes involved in the biosynthesis of the pterin ring structure. This suggests the presence of an unrecognized DHNTP pyrophosphohydrolase gene in bacteria. As humans do not harbor DHNTP pyrophosphohydrolase, the high structural diversity of enzymes responsible for a reaction in tetrahydrofolate biosynthesis may provide an advantage in designing inhibitors targeting a specific group of bacteria in the intestinal microbiota.

Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.

Characterization of substrate specificity and inhibitory mechanism of bile salt hydrolase from Lactobacillus reuteri CRL 1098 using molecular docking analysis.

OBJECTIVES: To elucidate the molecular mechanisms involved in the substrate interaction of the bile salt hydrolase of Lactobacillus reuteri CRL 1098 (LrBSH) with bile acids (BAs) and to evaluate potential enzyme inhibitors based on computer and in vitro modeling assays. RESULTS: Asp19, Asn79, and Asn171 participated in the LrBSH interaction with all BAs tested while Leu56 and Glu 222 played an important role in the interaction with glyco- and tauro-conjugated BAs, respectively. A great percentage of hydrophobic and polar interactions were responsible for the binding of LrBSH with glyco- and tauro-conjugated BAs, respectively. Remarkably, the four binding pocket loops participated in the substrate binding site of LrBSH unlike most of the reported BSHs. Inhibition assays showed that ascorbic acid, citric acid, penicillin G, and ciprofloxacin decreased LrBSH activity by 47.1%, 40.14%, 28.8%, and 9%, respectively. Docking analysis revealed that tetracycline and caffeic acid phenethyl ester had the low binding energy (-7.32 and -7.19 kcal/mol, respectively) and resembled the interaction pattern of GDCA (-6.88 kcal/mol) while penicillin (-6.25 kcal/mol) and ascorbic acid (-5.98 kcal/mol) interacted at a longer distance. CONCLUSION: This study helps to delve into the molecular mechanisms involved in the recognition of substrates and potential inhibitors of LrBSH.

Determining the optimum model parameters for oligosaccharide production efficiency using response surface integrated particle swarm optimization method: an experimental validation study.

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45 U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.

Structural characterization of mixed-linkage α-glucans produced by mutants of Lactobacillus reuteri TMW 1.106 dextransucrase.

Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.

Contribution of glutaminases to glutamine metabolism and acid resistance in Lactobacillus reuteri and other vertebrate host adapted lactobacilli.

The bacterial conversion of glutamine to glutamate is catalyzed by glutamine-amidotransferases or glutaminases. Glutamine deamination contributes to the formation of the bioactive metabolites glutamate, γ-aminobutyrate (GABA) and γ-glutamyl peptides, and to acid resistance. This study aimed to investigate the distribution of glutaminase(s) in lactobacilli, and to evaluate their contribution in L. reuteri to amino acid metabolism and acid resistance. Phylogenetic analysis of the glutaminases gls1, gls2 and gls3 in the genus Lactobacillus demonstrated that glutaminase is exclusively present in host-adapted species of lactobacilli. The disruption gls1, gls2 and gls3 in L. reuteri 100-23 had only a limited effect on the conversion of glutamine to glutamate, GABA, or γ-glutamyl peptides in sourdough. The disruption of all glutaminases in L. reuteri 100-23Δgls1Δgls2Δgls3 but not disruption of gls2 and gls3 eliminated the protective effect of glutamine on the survival of the strain at pH 2.5. Glutamine also enhanced acid resistance of L. reuteri 100-23ΔgadB and L. taiwanensis 107q, strains without glutamate decarboxylase activity. Taken together, the study demonstrates that glutaminases of lactobacilli do not contribute substantially to glutamine metabolism but enhance acid resistance. Their exclusive presence in host-adapted lactobacilli provides an additional link between the adaptation of lactobacilli to specific habitats and their functionality when used as probiotics and starter cultures.

Enzymatic Synthesis and Characterization of Mono-, Oligo-, and Polyglucosylated Conjugates of Caffeic Acid and Gallic Acid.

Glucansucrases can be used to glucosylate various plant-derived phenolic compounds by using sucrose as donor substrate. We applied Lactobacillus reuteri TMW 1.106 dextransucrase to glucosylate the acceptor substrates caffeic acid and gallic acid. Subsequently, monoglucosylated and in particular oligo- and polyglucosylated conjugates were characterized by using different chromatographic techniques and two-dimensional NMR spectroscopy. Both acceptors were substituted at positions O3 and O4. Under the conditions used, two monoglucosylated products were formed for caffeic acid, whereas only one O3-monosubstituted conjugate was detected for gallic acid. However, both acceptors resulted in O4-substituted oligo- and polyglucosylated conjugates, the amount of which was higher from gallic acid than from caffeic acid. Profile analysis tensiometry suggested that, in contrast to unmodified dextrans, oligo- and polymeric glucoconjugates of gallic acid are highly interfacially active. Overall, we provide the first detailed characterization of enzymatically conjugated oligo- and polymeric dextrans, which may have further potential as functional ingredients.

Biodetoxification of fungal mycotoxins zearalenone by engineered probiotic bacterium Lactobacillus reuteri with surface-displayed lactonohydrolase.

Zearalenone (ZEN) is one of the common mycotoxins with quite high occurrence rate and is harmful to animal and human health. Lactobacillus reuteri is known as a probiotic bacterium with active immune stimulating and high inhibitory activity against pathogenic microorganisms. In this study, we expressed the lactonohydrolase from Rhinocladiella mackenziei CBS 650.93 (RmZHD) in L. reuteri via secretion and surface-display patterns, respectively. Endogenous signal peptides from L. reuteri were first screened to achieve high expression for efficient ZEN hydrolysis. For secretion expression, signal peptide from collagen-binding protein showed the best performance, while the one from fructose-2,6-bisphosphatase worked best for surface-display expression. Both of the engineered strains could completely hydrolyze 5.0 mg/L ZEN in 8 h without detrimental effects on bacterial growth. The acid and bile tolerance assay and anchoring experiment on Caco-2 cells indicated both of the abovementioned engineered strains could survive during digestion and colonize on intestinal tract, in which the surface-displayed strain had a better performance on ZEN hydrolysis. Biodetoxification of model ZEN-contaminated maize kernels showed the surface-displayed L. reuteri strain could completely hydrolyze 2.5 mg/kg ZEN within 4 h under low water condition. The strain could also efficiently detoxify natural ZEN-contaminated corn flour in the in vitro digestion model system. The colonized property, survival capacity, and the efficient hydrolysis performance as well as probiotic functionality make L. reuteri strain an ideal host for detoxifying residual ZEN in vivo, which shows a great potential for application in feed industry.

Microbial production of medium chain fructooligosaccharides by recombinant yeast secreting bacterial inulosucrase.

A high yielding and straightforward production system of fructooligosaccharide (FOS) was developed for industrial production of prebiotics. To increase conversion yield of FOS from sucrose, recombinant yeast secreting inulosucrase from Lactobacillus reuteri (LrInu) were constructed. Efficient secretion of LrInu was achieved by truncation of both amino- and carboxy-termini (LrInuΔNC) and by introducing an optimal secretion signal. The recombinant yeast produced 220 U/mL of recombinant LrInuΔNC into culture medium during fed-batch fermentation. By direct fermentation of recombinant yeast in medium containing sucrose, 128.4 g/L of FOS was produced with 85.6% conversion yield from 300 g/L sucrose, and the highest titer was 152.6 g/L from 400 g/L sucrose. The degree of polymerization of generated FOS was 2-20 indicating medium chain (mcFOS) range. This is the first report of industrially applicable production of mcFOS by recombinant yeast secreting bacterial inulosucrase.

Temperature-dependent inulin nanoparticles synthesized by Lactobacillus reuteri 121 inulosucrase and complex formation with flavonoids.

Inulin nanoparticles (INNPs) are a biocompatible material which has a potential application for enhancing solubility and preventing degradation of compounds. In this work, we demonstrated that INNPs could be synthesized from sucrose using inulosucrase from Lactobacillus reuteri 121. Noticeably, dynamic light scattering (DLS) analysis showed that the derived INNPs exhibited uniformity in size, which was easily controlled by the reaction temperature. The effect of enzyme and sucrose concentration, as well as reaction time, was explored. Moreover, the solubility of INNPs in various organic solvents was also investigated, and we found that the INNPs were freely regenerated in water even though they had precipitated by organic solvents. Essentially, we demonstrated that the derived INNPs could be applied for flavonoid encapsulation. The solubility and stability of quercetin and fisetin in the INNPs complexes was higher than those of free compounds. These results make the INNPs very promising for many applications.

Glucosylation of flavonoids and flavonoid glycosides by mutant dextransucrase from Lactobacillus reuteri TMW 1.106.

Flavonoids are commonly abundant, plant-derived polyphenolic compounds which are responsible for color, taste, and antioxidant properties of certain plant based foods. Glucosylation by glucansucrases or other glycosyltransferases/glycoside hydrolases has been described to be a promising approach to modify stability, solubility, bioavailability, and taste profile of flavonoids and other compounds. In this study, we modified and applied a recombinant dextransucrase from Lactobacillus reuteri TMW 1.106 to glucosylate various flavonoids and flavonoid glycosides. The glucoconjugates were subsequently isolated and characterized by using two-dimensional NMR spectroscopy. Efficient glucosylation was achieved for quercetin and its glycosides quercetin-3-O-ß-glucoside and rutin. Significant portions of α-glucose conjugates were also obtained for epigallocatechin gallate, dihydromyricetin, and cyanidin-3-O-ß-glucoside, whereas glucosylation efficiency was low for naringin and neohesperidin dihydrochalcone. Most of the flavonoids with a catechol or pyrogallol group at the B-ring were predominantly glucosylated at position O4'. However, glycosyl substituents such as ß-glucose, rutinose, or neohesperidose were glucosylated at varying positions. Therefore, mutant dextransucrase from L. reuteri TMW 1.106 can be applied for versatile structural modification of flavonoids.

Lactobacillus mucosae DPC 6426 as a bile-modifying and immunomodulatory microbe.

BACKGROUND: Lactobacillus mucosae DPC 6426 has previously demonstrated potentially cardio-protective properties, in the form of dyslipidaemia and hypercholesterolemia correction in an apolipoprotein-E deficient mouse model. This study aims to characterise the manner in which this microbe may modulate host bile pool composition and immune response, in the context of cardiovascular disease. Lactobacillus mucosae DPC 6426 was assessed for bile salt hydrolase activity and specificity. The microbe was compared against several other enteric strains of the same species, as well as a confirmed bile salt hydrolase-active strain, Lactobacillus reuteri APC 2587. RESULTS: Quantitative bile salt hydrolase assays revealed that enzymatic extracts from Lactobacillus reuteri APC 2587 and Lactobacillus mucosae DPC 6426 demonstrate the greatest activity in vitro. Bile acid profiling of porcine and murine bile following incubation with Lactobacillus mucosae DPC 6426 confirmed a preference for hydrolysis of glyco-conjugated bile acids. In addition, the purified exopolysaccharide and secretome of Lactobacillus mucosae DPC 6426 were investigated for immunomodulatory capabilities using RAW264.7 macrophages. Gene expression data revealed that both fractions stimulated increases in interleukin-6 and interleukin-10 gene transcription in the murine macrophages, while the entire secretome was necessary to increase CD206 transcription. Moreover, the exopolysaccharide elicited a dose-dependent increase in nitric oxide and interleukin-10 production from RAW264.7 macrophages, concurrent with increased tumour necrosis factor-α secretion at all doses. CONCLUSIONS: This study indicates that Lactobacillus mucosae DPC 6426 modulates both bile pool composition and immune system tone in a manner which may contribute significantly to the previously identified cardio-protective phenotype.

Modulation of fructooligosaccharide chain length and insight into the product binding motif of Lactobacillus reuteri 121 inulosucrase.

Inulosucrase (E.C. 2.4.1.9) is a bacterial fructosyltransferase that synthesizes inulin-type fructooligosaccharide, using sucrose as a substrate. We modulated the size of fructooligosaccharide synthesized by Lactobacillus reuteri 121 inulosucrase using rational designed mutagenesis. Nine residues: D478, D479, S482, R483, N543, W551, N555, N561 and D689, were changed based on the active site architecture and amino acids potentially interacting with saccharides. The selected residues were substituted with alanine to investigate the contribution of these residues to FOS chain length. Enzymatic activity assays demonstrated that the transglycosylation/hydrolysis ratios of D479A, R483A, N543A, W551A and N555A mutants were significantly different from that of the wild type. Almost all mutants, except D478A, synthesized oligosaccharides with different size distribution compared to that of wild type. Molecular docking further provides insights into the product binding motif of Lactobacillus reuteri 121 inulosucrase and strengthens an important role of amino acid residues at remote locations from the active site on the enzymatic activity and product specificity.

Overcoming NADPH product inhibition improves D-sorbitol conversion to L-sorbose.

Gluconobacter oxydans sorbitol dehydrogenase (GoSLDH) exhibits a higher catalytic efficiency than other L-sorbose producing enzymes. During the reaction catalysed by GoSLDH, NADP+ is reduced to NADPH and D-sorbitol is oxidized to L-sorbose. However, GoSLDH activity is inhibited by the NADPH (Ki = 100 µM) formed during the enzymatic reaction. Therefore, Escherichia coligosldh-lrenox producing both GoSLDH for D-sorbitol oxidation and LreNOX (NAD(P)H oxidase from Lactobacillus reuteri) for NADP+ regeneration was generated and used for L-sorbose production. Whole cell biocatalysts with the LreNOX cofactor recycling system showed a high conversion rate (92%) of D-sorbitol to L-sorbose in the presence of low concentration of NADP+ (0.5 mM). By alleviating NADPH accumulation during the catalytic reactions, E. coligosldh-lrenox exhibited 23-fold higher conversion rate of D-sorbitol than E. coligosldh. L-Sorbose production by E. coligosldh-lrenox reached 4.1 g/L after 40 min, which was 20.5-fold higher than that of E. coligosldh. We also constructed G. oxydansgosldh and G. oxydansgosldh-lrenox strains, and they exhibited 1.2- and 2.9-fold higher conversion rates than the wild-type G. oxydans KCTC 1091. The results indicate that overcoming NADPH product inhibition using LreNOX improves chemical production in NADP+-dependent enzymatic reactions.

Lactobacillus reuteri NAD(P)H oxidase: Properties and coexpression with propanediol-utilization enzymes for enhancing 3-hydroxypropionic acid production from 3-hydroxypropionaldehyde.

Lactobacillus reuteri metabolizes glycerol through propanediol-utilization (Pdu) pathway to 1,3-propanediol (1,3-PD) via 3-hydroxypropionaldehyde (3-HPA) as intermediate. In the resting cells, the oxidized co-factor obtained in the reaction is regenerated by simultaneous oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP) using propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). We have earlier shown that the use of resting cells of recombinant Escherichia coli expressing the oxidative pathway gives the highest theoretical yield of 1 mol 3-HP per mol 3-HPA but is limited by cofactor depletion. In the present study, the gene encoding the enzyme NAD(P)H oxidase (LreuNox) that utilizes molecular oxygen as substrate, was isolated from L. reuteri and heterologously overexpressed in E. coli. LreuNox has a pH optimum of 6 and exhibits Vmax of 101.1 ± 2.2 U/mg with NADH, which is 30% higher than that for NADPH. Co-expression of LreuNox with PduP, PduL and PduW in E. coli enhances the biocatalytic lifetime as well as productivity at least two-fold compared to that achieved without co-factor regeneration.

Characterization of a 4,6­α­glucanotransferase from Lactobacillus reuteri E81 and production of malto-oligosaccharides with immune-modulatory roles.

A wide number of Lactic Acid Bacteria (LAB) species produce α-glucans with their ability to synthesize glucansucrases (GS) which use sucrose as substrate for the glucan production. Recently another group of enzymes in LAB gained special interest for their ability to produce α-glucans targeting the substrates containing α1-4-linkages and synthesizing new (α1-6) or (α1-3)-linkages as α­glucanotransferases. In this study, a putative 4,6­α­glucanotransferase (GTFB) from sourdough isolate Lactobacillus reuteri E81 was identified and expressed in Escherichia coli. The biochemical characterization of the GTFB-E81 confirmed its function as it cleaved the α1-4-linkages in different substrates and produced new gluco-oligomers/polymers containing α1-6 linkages together with the α1-4-linkages detected by NMR analysis. GTFB-E81 produced malto-oligosaccharides targeting maltose and maltoheptaose as substrates with up to DP 8 detected by TLC and ESI-MS/MS analysis. The functional roles of these malto-oligosaccharides were determined by testing their immune-modulatory functions in HT29 cells and they triggered the production of anti-inflammatory 1L-4 and pro-inflammatory IL-12 cytokines.

Structural characterization of glucosylated GOS derivatives synthesized by the Lactobacillus reuteri GtfA and Gtf180 glucansucrase enzymes.

ß-Galacto-oligosaccharides (GOS) are used commercially in infant nutrition, aiming to functionally replace human milk oligosaccharides (hMOS). Glucansucrases Gtf180-ΔN and GtfA-ΔN of Lactobacillus reuteri strains convert sucrose into α-glucans with (α1→6)/(α1→3) and (α1→4)/(α1→6) glucosidic linkages, respectively. Previously we reported that both glucansucrases glucosylate lactose, producing a minimum of 5 compounds (degree of polymerization 3-4) (GL34 mixture) with (α1→2/3/4) linkages. This GL34 mixture exhibited growth stimulatory effects on various probiotic bacteria. Aiming to obtain additional compounds mimicking hMOS in structure and function, we here studied glucosylation of 3 commercially available galactosyl-lactose GOS compounds. Both Gtf180-ΔN and GtfA-ΔN were unable to use 3'-galactosyl-lactose (ß3'-GL), but used sucrose to add a single glucose moiety to 4'-galactosyl-lactose (ß4'-GL) and 6'-galactosyl-lactose (ß6'-GL). ß6'-GL was elongated at its reducing glucosyl unit with an (α1→2)-linked moiety and at its non-reducing end with an (α1→4) linked moiety; ß4'-GL was only elongated at its reducing end with an (α1→2) linked moiety. Glucansucrases Gtf180-ΔN and GtfA-ΔN thus can be used to produce galactosyl-lactose-derived oligosaccharides containing (α1→2) and (α→4) glucosidic linkages, potentially with valuable bioactive (prebiotic) properties.

Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate.

Glucansucrase Gtf180-ΔN from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (α1→2)/(α1→3)/(α1→4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (α1→3) linkage and increased F4 4',2-glc-lac with (α1→4)/(α1→2) linkages. Formation of F2 2-glc-lac with an (α1→2) linkage and F4 was negatively affected in most W1065 and N1029 mutants, respectively. Mutant N1029G synthesized four new products with additional (α1→3)-linked glucosyl moieties (2xDP4 and 2xDP5). Sucrose/lactose strongly reduced Gtf180-ΔN hydrolytic activity and increased transferase activity of Gtf180-ΔN and mutant N1029G, in comparison to activity with sucrose alone. N1029/W1065/Q1140 thus are key determinants of Gtf180-ΔN linkage and product specificity in the acceptor reaction with lactose. Mutagenesis of key residues in Gtf180-ΔN may allow synthesis of tailor-made mixtures of novel lactose-derived oligosaccharides with potential applications as prebiotic compounds in food/feed and in pharmacy/medicine.

γ-Glutamyl Cysteine Ligase of Lactobacillus reuteri Synthesizes γ-Glutamyl Dipeptides in Sourdough.

Kokumi-active γ-glutamyl dipeptides (γ-GPs) accumulate in fermented food. γ-Glutamyl transferase, glutaminase, glutathione synthetase, and γ-glutamyl cysteine ligase (GCL) may synthesize γ-GPs. The genome of Lactobacillus reuteri encodes GCL but not glutathione synthetase or glutamyl transferase; therefore, this study investigated the role of GCL in γ-GP synthesis by L. reuteri LTH5448. Phylogenomic analysis of gcl in lactobacilli demonstrated that three genes coding for GCL are present in L. reuteri; two of these are present in L. reuteri LTH5448. Two deletion mutants of L. reuteri LTH5448, L. reuteri LTH5448Δ gcl1 and LTH5448Δ gcl1Δ gcl2, were constructed by double crossover mutagenesis. Growth and oxygen resistance of the mutants were comparable to the wild type. γ-Glu-Glu, γ-Glu-Leu, γ-Glu-Ile, γ-Glu-Val, and γ-Glu-Cys were quantified in buffer and sourdough fermentations by liquid chromatography-mass spectrometry. The wild type and L. reuteri Δ gcl1 but not Δ gcl1Δ gcl2 converted amino acids to γ-Glu-Cys. γ-Glu-Ile accumulation was reduced in both mutants; however, the disruption of gcl did not alter the biosynthesis of the other γ-GPs. In conclusion, gcl1 in L. reuteri mediates γ-Glu-Ile synthesis, gcl2 mediates γ-Glu-Cys synthesis, but neither gene affected synthesis of other γ-GPs. This study facilitates selection of starter cultures that synthesize γ-Glu peptides with kokumi activity and, thus, improve the taste of fermented foods.

Site-specific hydrolysis of chlorogenic acids by selected Lactobacillus species.

Hydroxycinnamic acids are a major group of phenolic compounds widely distributed in plants. Among them, chlorogenic acids and caffeic acid have been in the focus of interest due to their impact on food quality and their putative health benefits. Numerous microorganisms like lactic acid bacteria are able to hydrolyze chlorogenic acids by cinnamoyl esterase enzymes. Data on the specificity of theses enzymes regarding the cleavage of distinct isomers of mono- or dichlorogenic acids is lacking. Lactobacillus reuteri, Lactobacillus helveticus, and Lactobacillus fermentum were screened for their ability to hydrolyze chlorogenic acid isomers in culture medium. Concentrations of chlorogenic acids and the released caffeic acid were determined by UHPLC-ESI-MS. The highest hydrolysis rate (100%) was observed for the hydrolysis of 5-CQA by Lactobacillus helveticus. A so far unknown metabolic pathway for the cleavage of 4-CQA is proposed including isomerization to 5-CQA and 3-CQA followed by hydrolysis.