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Even though milk oligosaccharides are not digested and absorbed in the small intestine in most eutherian young, the milk of some eutherian species, including humans, still contains milk oligosaccharides as well as the predominant lactose. Keywords: -lactalbumin; colonic bifidobacteria; lactose; lysozyme; milk oligosaccharides; small intestinal lactase EN -lactalbumin colonic bifidobacteria lactose lysozyme milk oligosaccharides small intestinal lactase 14 23 10 06/20/23 20230601 NES 230601 Implications -Lactalbumin, a milk protein, had evolved from c-type lysozyme, and it can associate with 4-galactosyltransferase 1 within the mammary epithelial cells. The predominance of lactose or milk oligosaccharides in the carbohydrate fractions of milk should be controlled by the rate of lactose and milk oligosaccharide biosynthesis. [Extracted from the article]

Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189 kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity. Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose [ABSTRACT FROM AUTHOR]

Polyphenol oxidases (PPOs) catalyze browning reactions in various plant organs, therefore controlling the reactions is important for the food industry. PPOs have been assumed to be involved in skin browning of white grape cultivars; however, the molecular mechanism underlying PPO-mediated browning process remains elusive. We have recently identified a newPPOgene namedVvPPO2from “Shine Muscat” (Vitis labruscanaBailey × V. viniferaL.), and have shown that the gene is transcribed at a higher level than the previously identifiedVvPPO1in browning, physiologically disordered berry skins at the maturation stage. In this study, we expressedVvPPO2 inEscherichia coliand, using the purified preparation, revealed unique physicochemical characteristics of the enzyme. Our study opens up a way to not only understand the berry skin browning process but also to elucidate the enzymatic maturation process of grape PPOs. Characterization of Polyphenol Oxidase 2 (VvPPO2) from ‘Shine Muscat’ (Vitis labruscanaBailey ×Vitis viniferaL.) [ABSTRACT FROM AUTHOR]

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on theO-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin typeO-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of severalBifidobacteriumstrains to degrade a sulfated glycan substrate and identified a 6-sulfo-β-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded bybbhIIfromBifidobacterium bifidumJCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfatedN-acetylglucosamines over non-sulfated and 3-sulfatedN-acetylglucosamines. The purified BbhII directly released 6-sulfatedN-acetylglucosamine from porcine gastric mucin and the expression ofbbhIIwas moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes. BbhII encodes a sulfoglycosidase which is involved in the mucin degradation pathway ofBifidobacterium bifidum. [ABSTRACT FROM PUBLISHER]

Recently, a “human gut microbial gene catalogue,” which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the “human gut microbial gene catalogue” were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based onin silicogenomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest. The cultivability in GAM of 44 of the top 56 species listed in the “human gut microbial gene catalogue” was tested; of these, 32 (72%) were successfully cultured. [ABSTRACT FROM PUBLISHER]

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase fromBifidobacterium bifidumJCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and inO-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues intoN- andO-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics. Introduction of H-antigen structure (Fucα1-2Gal) into various glycoconjugates by 1,2-α-l-fucosynthase. [ABSTRACT FROM PUBLISHER]

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine usingEscherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combiningin vivotetrahydropapaveroline production inE. coliwithin vitroenzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery. High amount of pure (S)-reticuline. [ABSTRACT FROM AUTHOR]

Sake is made from steamed rice, malted rice, and water. Sake production begins with the preparation of a small-scale starter (moto); the quality ofmotosignificantly influences the flavor and richness of sake. In the traditional starter,yamahai-moto, the growth of naturally occurring lactic acid bacteria represses the putrefactive micro-organisms, whereas in the modern starter,sokujo-moto, this is achieved by adding lactic acid. In this study, the successive change in bacterial flora ofyamahai-motowas analyzed by pyrosequencing 16S ribosomal RNA genes.Lactobacilluswas dominant throughout the process (93–98%). Nitrate-reducing bacteria that have been generally assumed to be the first colonizers ofyamahai-motowere scarcely found in the early stage, butLactobacillus acidipiscisdominated.Lactobacillus sakeidrastically increased in the middle stage. This is the first report, though one case study, to show how the early stage microbiota in Japaneseyamahai-motois varyingly controlled without nitrate-reducing bacteria using next-generation sequencing. Microbiota change during the fermentation of traditional Japanese sake starteryamahai-moto. [ABSTRACT FROM PUBLISHER]

Glycoside hydrolase family 42 (GH42) includes β-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different β-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite −1 to be key specificity determinants. Bga42A thus prefers the β1-3-galactosidic linkage from human milk and other β1-3- and β1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galβ1-4Galβ1-4Glc) as well as 4-galactobiose (Galβ1-4Gal) and 4-galactotriose (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside-rich gut niche and emphasize the importance and diversity of β-galactoside metabolism in probiotic bifidobacteria. [ABSTRACT FROM PUBLISHER]

Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22:361–368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 α-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed α1,3-linked Gal in branched blood group B antigen [Galα1-3(Fucα1-2)Galβ1-R], but not in a linear xenotransplantation antigen (Galα1-3Galβ1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts. [ABSTRACT FROM PUBLISHER]

The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized β-galactosidases of this subspecies to understand how the organism degrades type-1 (Galβ1-3GlcNAc) and type-2 (Galβ1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel β-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Galβ1-3GlcNAcβ1-3Galβ1-4Glc) than for lacto-N-biose I (Galβ1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT β-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a β-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription–polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different β-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of β-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans. [ABSTRACT FROM PUBLISHER]

The article presents the findings of a study conducted on examining the changes in the microbial population in aji (horse mackerel)-narezushi during fermentation by pyrosequencing the 16S ribosomal RNA gene (rDNA). It mentions that lactobacillus population drastically increased during fermentation, while the populations of other bacteria remained unchanged. It concludes that high product-dependency of the bacterial composition might be due to the starter-free fermentation process.

Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different α-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-α-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-α-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50°C. Notably, 80% activity remained after 30 min incubation at 80°C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the α2,3-linkage rather than α2,6- and α2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-α-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs. [ABSTRACT FROM AUTHOR]

Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc; type 1 chain) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one β-galactosidase (BbgIII) and two β-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto-N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs. [ABSTRACT FROM PUBLISHER]

Endo-α-N-acetylgalactosaminidase (endo-α-GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyses the O-glycosidic bonds in mucin-type O-glycan between α-GalNAc and Ser/Thr. Endo-α-GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Galβ1-3GalNAc (GNB), whereas endo-α-GalNAc-ase from Clostridium perfringens (EngCP) exhibits broader substrate specificity. We determined the crystal structure of EngBF at 2.0 Å resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the β-faces of sugar rings of GNB by substrate-induced fit. The difference in substrate specificities between EngBF and EngCP is attributed to the variations in amino acid sequences in the regions forming the substrate-binding pocket. Our results provide a structural basis for substrate recognition by GH101 endo-α-GalNAc-ases and will help structure-based engineering of these enzymes to produce various kinds of neo-glycoconjugates. [ABSTRACT FROM PUBLISHER]

Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-α-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-α-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885–4893). Here, we identified a gene encoding a novel 1,3–1,4-α-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 α-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released α1,3- and α1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing α1,2-fucosyl residue or on synthetic α-fucoside (p-nitrophenyl-α-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic α-l-fucosidase. The transformant carrying afcA could utilize 2′-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading α1,2- and α1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts. [ABSTRACT FROM PUBLISHER]

The article presents a study which determines whether zwitterionic glycosphingolipids (ZGLs) from the phytopathogenic filamentous fungus Trichoderma viride have functional roles in plant-fungus interactions. It notes that ZGLs were tested to identify if they would induce defense responses in cultured rice cells. It concludes that T. viride's ZGLs elicited expression of the PAL and PBZ1 genes, which are associated with pathogen resistance.

The article discusses a study which described the method of constructing a mutated plasmid library free from ligation. It is focused on the QuickChange site-directed mutagenesis methodology which was applied by adding manganese to the reaction mixture. It is described that the method is superior to the normally employed Pol I-type polymerase-based error-prone polymerase chain reaction. The method offered a construction of randomly multiage plasmid library with a free ligation reaction.

The growth of Escherichia coli is inhibited by an antibiotic compound, azaserine (O-diazoacetyl-l-serine). Previous studies revealed the biochemical properties of azaserine, which involves inhibition of various enzymatic reactions as well as introduction of DNA breakage. However, genetically, nothing has been elucidated except that all the azaserine-resistant strains isolated so far carry lesions in the aroP gene as a primary determinant. Here, we demonstrate that, in addition to AroP, the LIV-I/LS system, an ATP-binding cassette type transporter, is involved in azaserine sensitivity of E. coli, by genetic analysis and transport studies, in which Ki value for azaserine was determined to be ∼10-3 M. [Copyright &y& Elsevier]

The article discusses differences in the substrate specificities and active site structure of two-α-L-fucosidases enzymes present in Bacteroides thetaiotaomicron. It reports that α-L-fucosidases enzyme belongs to hydrolase family and is divided into two subfamilies based on phylogenetic clustering. It mentions that differences in structure of the enzyme were examined through analysis of various substrates at fixed concentration.

The article discusses the enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using Erwinia herbicola cells that carry the mutant transciptional regulator TyrR. It notes that L-dopa is used in the treating individuals with Parkinson's disease and is synthesized through enzymatic method that uses tyrosine phenol-lyase (Tp)l, a tyrosine-inducible enzyme. It notes that the highest productivity of L-dopa was obtained for the strain that was developed under tyrosine non-induced conditions.