Biotransformation of camu-camu galloylated ellagitannins by Lactiplantibacillus plantarum with extracellular tannase activity.

Some strains of Lactiplantibacillus plantarum produce specific tannases that could enable the metabolism of ellagitannins into more bioavailable phenolic metabolites, thereby promoting the health effects of these polyphenols. However, the metabolic ability of these strains remains poorly understood. In this study, we analyzed the ability of broad esterase-producing (Est_1092+) and extracellular tannase-producing (TanA+) strains to convert a wide assortment of ellagitannins from camu-camu (Myrciaria dubia) fruit. To this end, forty-three strains were screened to identify and sequence (WGS) those producing Est_1092. In addition, six previously reported TanA+ strains were included in the study. Each strain (Est_1092+ or TanA+) was inoculated into a minimal culture medium supplemented with an aqueous camu-camu extract. After fermentation, supernatants were collected for semi-quantification of ellagitannins and their metabolites by mass spectrometry. For analysis, the strains were grouped according to their enzyme type and compared with an Est_1092 and TanA-lacking strain. Out of the forty-three isolates, three showed Est_1092 activity. Of the Est_1092+ and TanA+ strains, only the latter hydrolyzed the tri-galloyl-HHDP-glucose and various isomers of HHDP-galloyl-glucose, releasing HHDP-glucose and gallic acid. TanA+ strains also transformed three isomers of di-HHDP-galloyl-glucose, liberating di-HHDP-glucose and gallic acid. Overall, TanA+ strains released 3.6-4.9 times more gallic acid than the lacking strain. In addition, those exhibiting gallate decarboxylase activity pursued gallic acid metabolism to release pyrogallol. Neither Est_1092+ nor TanA+ strains transformed ellagitannin-core structures. In summary, TanA+ L. plantarum strains have the unique ability to hydrolyze a wide range of galloylated ellagitannins, releasing phenolic metabolites with additional health benefits.

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

pH-dependent regulation of an acidophilic O-acetylhomoserine sulfhydrylase from Lactobacillus plantarum.

Bacteria have two routes for the l-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated l-homoserine is directly converted into l-homocysteine. The reaction using H2S as the second substrate is catalyzed by a pyridoxal 5'-phosphate-dependent enzyme, O-acetylhomoserine sulfhydrylase (OAHS). In the present study, we determined the enzymatic functions and the structures of OAHS from Lactobacillus plantarum (LpOAHS). The LpOAHS enzyme exhibited the highest catalytic activity under the weak acidic pH condition. In addition, crystallographic analysis revealed that the enzyme takes two distinct structures, open and closed forms. In the closed form, two acidic residues are sterically clustered. The proximity may cause the electrostatic repulsion, inhibiting the formation of the closed form under the neutral to the basic pH conditions. We concluded that the pH-dependent regulation mechanism using the two acidic residues contributes to the acidophilic feature of the enzyme. IMPORTANCE: In the present study, we can elucidate the pH-dependent regulation mechanism of the acidophilic OAHS. The acidophilic feature of the enzyme is caused by the introduction of an acidic residue to the neighborhood of the key acidic residue acting as a switch for the structural interconversion. The strategy may be useful in the field of protein engineering to change the optimal pH of the enzymes. In addition, this study may be useful for the development of antibacterial drugs because the l-methionine synthesis essential for bacteria is inhibited by the OAHS inhibitors. The compounds that can inhibit the interconversion between the open and closed forms of OAHS may become antibacterial drugs.

Heterologous expression and enzymatic characteristics of sulfatase from Lactobacillus plantarum dy-1.

Barley, rich in bioactive components including dietary fiber, polyphenolic compounds and functional proteins, exhibits health benefits such as regulating glucose and lipid metabolism. Previous studies have found that the content and composition of free phenolic acids in barley may be significantly changed by fermentation with the laboratory patented strain Lactobacillus plantarum dy-1 (L. p dy-1), but the mechanism of enzymatic release of phenolic acid remains to be elucidated. Based on this, this study aimed to identify the key enzyme in L. p dy-1 responsible for releasing the bound phenolic acid and to further analyze its enzymatic properties. The Carbohydrate-Active enZYmes database revealed that L. p dy-1 encodes 7 types of auxiliary enzymes, among which we have identified a membrane sulfatase. The enzyme gene LPMS05445 was heterologous to that expressed in E. coli, and a recombinant strain was induced to produce the target protein and purified. The molecular weight of the purified enzyme was about 59.9 kDa, with 578.21 U mg-1 enzyme activity. The optimal temperature and pH for LPMS05445 expression were 40 °C and 7.0, respectively. Furthermore, enzymatic hydrolysis by LPMS05445 can obviously change the surface microstructure of dietary fiber from barley bran and enhance the release of bound phenolic acid, thereby increasing the free phenolic acid content and improving its physiological function. In conclusion, sulfatase produced by Lactobacillus plantarum dy-1 plays a key role in releasing bound phenolic acids during the fermentation of barley.

Production of prebiotic enriched maple syrup through enzymatic conversion of sucrose into fructo-oligosaccharides.

Maple syrup, a popular natural sweetener has a high content of sucrose, whose consumption is linked to different health issues such as obesity and diabetes. Hence, within this paper, the conversion of sucrose to prebiotics (fructo-oligosaccharides, FOS) was proposed as a promising approach to obtaining a healthier, value-added product. Enzymatic conversion was optimized with respect to key experimental factors, and thereafter derived immobilized preparation of fructosyltransferase (FTase) from Pectinex® Ultra SP-L (FTase-epoxy Purolite, 255 IU/g support) was successfully utilized to produce novel functional product in ten consecutive reaction cycles. The product, obtained under optimal conditions (60 °C, 7.65 IU/mL, 12 h), resulted in 56.0% FOS, 16.7% sucrose, and 27.3% monosaccharides of total carbohydrates, leading to a 1.6-fold reduction in caloric content. The obtained products` prebiotic potential toward the probiotic strain Lactobacillus plantarum 299v was demonstrated. The changes in physico-chemical and sensorial characteristics were esteemed as negligible.

A comparative study on flaxseed lignan biotransformation through resting cell catalysis and microbial fermentation by ß-glucosidase production Lactiplantibacillus plantarum.

BACKGROUND: Flax lignan has attracted much attention because of its potential bioactivities. However, the bioavailability of secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, depends on the bioconversion by the colon bacteria. Lactic acid bacteria (LAB) with ß-glucosidase activity has found wide application in preparing bioactive aglycone. RESULTS: LAB strains with good ß-glucosidase activity were isolated from fermented tofu. Their bioconversion of flax lignan extract was investigated by resting cell catalysis and microbial fermentation, and the metabolism of SDG by Lactiplantibacillus plantarum C5 following fermentation was characterized by widely targeted metabolomics. Five L. plantarum strains producing ß-glucosidase with broad substrate specificity were isolated and identified, and they all can transform SDG into secoisolariciresinol (SECO). L. plantarum C5 resting cell reached a maximum SDG conversion of 49.19 ± 3.75%, and SECO generation of 21.49 ± 1.32% (0.215 ± 0.013 mm) at an SDG substrate concentration of 1 mM and 0.477 ± 0.003 mm SECO was produced at 4 mm within 24 h. Although sixteen flax lignan metabolites were identified following the fermentation of SDG extract by L. plantarum C5, among them, four were produced following the fermentation: SECO, demethyl-SECO, demethyl-dehydroxy-SECO and isolariciresinol. Moreover, seven lignans increased significantly. CONCLUSION: Fermentation significantly increased the profile and level of flax lignan metabolites, and the resting cell catalysis benefits from higher bioconversion efficiency and more straightforward product separation. Resting cell catalysis and microbial fermentation of flax lignan extract by the isolated ß-glucosidase production L. plantarum could be potentially applied in preparing flax lignan ingredients and fermented flaxseed. © 2024 Society of Chemical Industry.

Investigation of the Lethal Effect of Purified Arginine Deiminase Purified from Lactobacillus plantarum p5 on Murine Mammary Adenocarcinoma and Vero cell Lines.

Cancer is one of the most serious diseases facing humanity; accordingly, it is urgent to find a cure that is rarely harmful to the patient as much as possible. It has been approved that arginine deiminase (ADI) can hydrolyze the plasma arginine to citrulline. This hydrolysis activity and reduction in the amount of intercellular arginine suppress lipopolysaccharide-induced nitric oxide synthesis. On the other hand, arginine depletion arrests the cell cycle at the G1 phase; therefore, ADI has been considered a powerful anticancer agent. The current study aimed to investigate the lethal effects of ADI purified from the Lactobacillus plantarum p5 strain on murine mammary adenocarcinoma and Vero cell lines. Anti-proliferative activity of ADI against murine mammary adenocarcinoma) AMN3) cell line was evaluated after different incubation times (3, 6, 24, 48, and 72 h) of exposure to 1 µg/mL of ADI, compared to Vero (non-cancer cell line) transformed cell line with same conditions. The autophagy process in cancer cells was recognized after three hours of incubation with ADI which was clearly observed in the AMN3 cell line under an inverted microscope. The first stages of the programmed cell death (apoptosis) pathway were only observed in AMN3 cells after 24 h of incubation with ADI, and this process continued with the time until they reached the last stages of apoptosis after 72 h of incubation. The results of the current study showed that the AMN3 cell line was auxotrophic for arginine because it could not produce it in the presence of enzyme which had a robust activity to kill these cancer cells; however, Vero non-cancer cell line survived in the presence of ADI because it had the ability to produce arginine.

Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

Genes encoding bile salt hydrolase differentially affect adhesion of Lactiplantibacillus plantarum AR113.

BACKGROUND: Adhesion is considered important for Lactiplantibacillus to persist in the human gut and for it to exert probiotic effects. Lactiplantibacillus plantarum contains a considerable number and variety of genes encoding bile salt hydrolases (bsh), but their effects on microbial adhesion remain poorly understood. To clarify the effects of four bsh on adhesion, we tried to knock out bsh (Δbsh) of L. plantarum AR113 using the CRISPR-Cas9 method, and compared the growth, auto-aggregation (RAA ), co-aggregation (RCA ), surface hydrophobicity (AHC ) of AR113 wild-type and Δbsh strains and their adhesion abilities to HT29 cells. RESULTS: We first obtained the AR113 Δbsh1,3,2,4 strain with four bsh knocked out. Their growth was significantly slower than the wild-type strain cultured in De Man, Rogosa, and Sharpe medium (MRS) with 3.0 g L-1 glyco- or tauro-conjugated bile acid. Bsh had no significant effect on the growth of ten strains cultured in MRS, but Δbsh1 inhibited their growth when cultured in MRS containing 3.0 g L-1 sodium glycocholate, whereas Δbsh4 instead promoted their growth in MRS with 3.0 g L-1 sodium glycocholate and sodium taurocholate. RCA and RAA were linearly positive for all strains except AR113 Δbsh2,4, and AHC and RAA were negatively correlated for most strains excluding AR113 Δbsh2, with RAA  = 6.38-25.05%, RCA  = 5.17-9.22%, and ACH  = 3.22-47.71%. The adhesion ability of ten strains cultured in MRS was higher than that of strains cultured in MRS with 3.0 g L-1 bovine bile, and it was related to bsh2. CONCLUSION: Bsh differentially affected the adhesion of AR113 series strains. This adds to the available information about substrate-gene-performance, and provides new information to enable engineering to regulate the colonization of Lactiplantibacillus. © 2021 Society of Chemical Industry.

Characterization of a lactic acid bacterium-derived ß-glucosidase for the production of rubusoside from stevioside.

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5-6.5 and 30-40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher ß-glucosidase activity toward the ß-linked glucosidic bond of stevioside than toward other ß-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The ß-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this ß-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product.

Production of α-rhamnosidases from Lactobacillus plantarum WCFS1 and their role in deglycosylation of dietary flavonoids naringin and rutin.

This work addresses the amino acid sequence, structural analysis, biochemical characterization and glycosidase activity of two recombinant α-rhamnosidases, Ram1 and Ram2, from Lactobacillus plantarum WCFS1. The substrate specificity of both enzymes towards the disaccharide rutinose and natural dietary flavonoids naringin and rutin was also determined and compared to that of a commercial multienzyme complex (Pectinex Ultra Passover, PPO). Ram1 is a less acidic- and heat-active enzyme than Ram2 and exhibited a high activity towards pNP-α-L-rhamnopyranoside, but it was unable to hydrolyze neither rutinose, naringin or rutin. In contrast, Ram2 enzyme showed a substrate specificity towards α-(1➔6) glycosidic flavonoids, such as rutin, and the disaccharide rutinose. The mechanism of action of Ram2 towards rutin was elucidated and revealed the potential cost-effective and selective production of the monoglycosylated flavonoid isoquercetin (quercetin-3-O-glucoside). PPO efficiently converted both naringin and rutin into their corresponding aglycones. These findings revealed the potential usefulness of PPO for the improvement of sensory properties of beverages through debittering of citrus juices, as well as the potential use of Ram2 to selectively produce isoquercetin, a highly valued and bioactive flavonoid whose production is not currently affordable.

The LarB carboxylase/hydrolase forms a transient cysteinyl-pyridine intermediate during nickel-pincer nucleotide cofactor biosynthesis.

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.

Food-grade γ-aminobutyric acid production by immobilized glutamate decarboxylase from Lactobacillus plantarum in rice vinegar and monosodium glutamate system.

OBJECTIVES: γ-amino butyric acid (GABA) is a non-protein amino acid, considered a potent bioactive compound. This study focused on biosynthesis of food-grade GABA by immobilized glutamate decarboxylase (GAD) from Lactobacillus plantarum in the rice vinegar and monosodium glutamate (MSG) reaction system. RESULTS: The gene encoding glutamate decarboxylase (GadB) from L. plantarum has been heterologously expressed in Lactococcus lactis and biochemically characterized. Recombinant GadB existed as a homodimer, and displayed maximal activity at 40 °C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of GadB for L-Glu was 22.33 mM and 62.4 mM-1 min-1, respectively, with a specific activity of 24.97 U/mg protein. Then, purified GadB was encapsulated in gellan gum beads. Compared to the free enzyme, immobilized GadB showed higher operational and storage stability. Finally, 9.82 to 21.48 g/L of GABA have been acquired by regulating the amounts of catalyst microspheres ranging from 0.5 to 0.8 g (wet weight) in 0.8 mL of the designed rice vinegar and MSG reaction system. CONCLUSIONS: The method of production GABA by immobilized GadB microspheres mixed in the rice vinegar and MSG reaction system is introduced herein for the first time. Especially, the results obtained here meet the increased interest in the harnessing of biocatalyst to synthesize food-grade GABA.

A reverse catalytic triad Asp containing loop shaping a wide substrate binding pocket of a feruloyl esterase from Lactobacillus plantarum.

Feruloyl esterase is an indispensable biocatalyst in food processing, pesticide and pharmaceutical industries, catalyzing the cleavage of the ester bond cross-linked between the polysaccharide side chain of hemicellulose and ferulic acid in plant cell walls. LP_0796 from Lactobacillus plantarum was identified as a feruloyl esterase that may have potential applications in the food industry, but the lack of the substrate recognition and catalytic mechanisms limits its application. Here, LP_0796 showed the highest activity towards methyl caffeate at pH 6.6 and 40 °C. The crystal structure of LP_0796 was determined at 2.5 Å resolution and featured a catalytic triad Asp195-containing loop facing the opposite direction, thus forming a wider substrate binding pocket. Molecular docking simulation and site-directed mutagenesis studies further demonstrated that in addition to the catalytic triad (Ser94, Asp195, His225), Arg125 and Val128 played essential roles in the function of the active site. Our data also showed that Asp mutation of Ala23 and Ile198 increased the catalytic efficiency to 4- and 5-fold, respectively. Collectively, this work provided a better understanding of the substrate recognition and catalytic mechanisms of LP_0796 and may facilitate the future protein design of this important feruloyl esterase.

The use of Lactobacillus plantarum esterase genes: a biotechnological strategy to increase the bioavailability of dietary phenolic compounds in lactic acid bacteria.

In Lactobacillus plantarum the metabolism of hydroxybenzoic and hydroxycinnamic acid derivatives follows a similar two-step pathway, an esterase action followed by a decarboxylation. The L. plantarum esterase genes involved in these reactions have been cloned into pNZ8048 or pT1NX plasmids and transformed into technologically relevant lactic acid bacteria. None of the strains assayed can hydrolyse methyl gallate, a hydroxybenzoic ester. The presence of the L. plantarum tannase encoding genes (tanALp or tanBLp) on these bacteria conferred their detectable esterase (tannase) activity. Similarly, on hydroxycinnamic compounds, esterase activity for the hydrolysis of ferulic acid was acquired by lactic acid bacteria when L. plantarum esterase (JDM1_1092) was present. This study showed that the heterologous expression of L. plantarum esterase genes involved in the metabolism of phenolic acids allowed the production of healthy compounds which increase the bioavailability of these dietary compounds in food relevant lactic acid bacteria.

Dietary Inulin Increases Lactiplantibacillus plantarum Strain Lp900 Persistence in Rats Depending on the Dietary-Calcium Level.

Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit health benefits in the consumer. Lactiplantibacillus plantarum strains display high survival during transit through the mammalian gastrointestinal tract and were shown to have health-promoting properties. Growth on the fructose polysaccharide inulin is relatively uncommon in L. plantarum, and in this study we describe FosE, a plasmid-encoded ß-fructosidase of L. plantarum strain Lp900 which has inulin-hydrolyzing properties. FosE contains an LPxTG-like motif involved in sortase-dependent cell wall anchoring but is also (partially) released in the culture supernatant. In addition, we examined the effect of diet supplementation with inulin on the intestinal persistence of Lp900 in adult male Wistar rats in diets with distinct calcium levels. Inulin supplementation in high-dietary-calcium diets significantly increased the intestinal persistence of L. plantarum Lp900, whereas this effect was not observed upon inulin supplementation of the low-calcium diet. Moreover, intestinal persistence of L. plantarum Lp900 was determined when provided as a probiotic (by itself) or as a synbiotic (i.e., in an inulin suspension) in rats that were fed unsupplemented diets containing the different calcium levels, revealing that the synbiotic administration increased bacterial survival and led to higher abundance of L. plantarum Lp900 in rats, particularly in a low-calcium-diet context. Our findings demonstrate that inulin supplementation can significantly enhance the intestinal delivery of L. plantarum Lp900 but that this effect strongly depends on calcium levels in the diet.IMPORTANCE Synbiotics combine probiotics with prebiotics to synergistically elicit a health benefit in the consumer. Previous studies have shown that prebiotics can selectively stimulate the growth in the intestine of specific bacterial strains. In synbiotic supplementations the prebiotics constituent could increase the intestinal persistence and survival of accompanying probiotic strain(s) and/or modulate the endogenous host microbiota to contribute to the synergistic enhancement of the health-promoting effects of the synbiotic constituents. Our study establishes a profound effect of dietary-calcium-dependent inulin supplementation on the intestinal persistence of inulin-utilizing L. plantarum Lp900 in rats. We also show that in rats on a low-dietary-calcium regime, the survival and intestinal abundance of L. plantarum Lp900 are significantly increased by administering it as an inulin-containing synbiotic. This study demonstrates that prebiotics can enhance the intestinal delivery of specific probiotics and that the prebiotic effect is profoundly influenced by the calcium content of the diet.

Biosynthesis of Nondigestible Galactose-Containing Hetero-oligosaccharides by Lactobacillus plantarum WCFS1 MelA α-Galactosidase.

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with ß-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients.

Purification of the phytase enzyme from Lactobacillus plantarum: The effect on pansy growth and macro-micro element content.

In the present study, the phytase enzyme was purified from Lactobacillus plantarum with a 3.08% recovery, 9.57-purification fold, and with a specific activity of 278.82 EU/mg protein. Then, the effects of the 5 EU and 10 EU purified phytase was determined on the plant growth, quality, the macro-micro nutrient content of pansy (Viola × wittrockiana), which is of great importance in ornamental plants industry. The research was established under greenhouse conditions with natural light in 2017. The pansy seeds were coated with phytase enzyme solution, sown in a peat environment, and transferred to pots at the seedling period. In general, the 5 EU and 10 EU applications increase plant height, the number of leaves per plant, the number of side branches per plant, and flower height parameters compared to control. Also, micro- and macronutrient values in soil and plant samples were examined. According to the results, the phytase application on pansy cultivation positively affected the properties and yielded high quality of plants.

Catalytic specificity of the Lactobacillus plantarum cystathionine γ-lyase presumed by the crystallographic analysis.

The reverse transsulfuration pathway, which is composed of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL), plays a role to synthesize L-cysteine using L-serine and the sulfur atom in L-methionine. A plant-derived lactic acid bacterium Lactobacillus plantarum SN35N has been previously found to harbor the gene cluster encoding the CBS- and CGL-like enzymes. In addition, it has been demonstrated that the L. plantarum CBS can synthesize cystathionine from O-acetyl-L-serine and L-homocysteine. The aim of this study is to characterize the enzymatic functions of the L. plantarum CGL. We have found that the enzyme has the high γ-lyase activity toward cystathionine to generate L-cysteine, together with the ß-lyase activity toward L-cystine to generate L-cysteine persulfide. By the crystallographic analysis of the inactive CGL K194A mutant complexed with cystathionine, we have found the residues which recognize the distal amino and carboxyl groups of cystathionine or L-cystine. The PLP-bound substrates at the active site may take either the binding pose for the γ- or ß-elimination reaction, with the former being the major reaction in the case of cystathionine.

An efficient ABC transporter signal peptide directs heterologous protein secretion in food-grade hosts.

An efficient expression-secretion system for heterologous protein production in food-grade hosts, Lactobacillus plantarum and Bacillus subtilis, is still required to broaden their applications. The optimal signal peptide compatible with both the desired protein and the target host is important for the system. Here, we constructed new expression-secretion vectors to be used in both bacteria. A natural plasmid originating from food-grade L. plantarum BCC9546 was used as a core vector combined with a strong constitutive promoter, L-ldh promoter, and various signal peptides from several types of L. plantarum proteins: ABC transporter, cell wall-associated and extracellular proteins. A gene encoding 88-kDa amylase isolated from starch-related L. plantarum TBRC470 was used as a gene model to evaluate the systems. By comparing the amounts of secreted amylase from the recombinant strains to that of wild type, all signal peptides gave higher yields of secreted amylase in recombinant B. subtilis. Interestingly, two ABC transporter signal peptides from glutamine and mannose ABC transporters provided noticeably high levels of secreted amylase in recombinant L. plantarum. Moreover, these signal peptides also gave high yields of secreted amylase in recombinant B. subtilis. From the results, the signal peptide of glutamine ABC transporter, which functions in essential amino acid transportation that is a precursor for synthesis of nitrogen-containing compounds and nitrogen homeostasis, has a potential use in development of an efficient expression-secretion system for heterologous protein production in both food-grade hosts.