In vitro micropropagation of Piper longum – an important medicinal plant

Efficient and rapid tissue culture systems were developed for Piper longum, an important medicinal plant, through shoot tip multiplication and direct regeneration. Multiple shoots were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44–22.19 M benzyladenine (BA) and 4.64–13.9 M kinetin (K). Maximum number of shoots were induced with 8.9 M BA and 4.64 M K. Adventitious shoot regeneration from leaf segments was achieved on MS containing 3.6–22.19 M BA along with 3.31–12.4 M picloram (P). Shoot differentiation occurred directly from the leaf bases without intermediale callus formation. Maximum shoot buds were obtained on MS medium with 17.76 M BA and 8.28 M P. Elongated shoots were separated and rooted in MS supplemented with 2.46 M indole butyric acid (IBA). Plantlets, thus developed were established in soil.     

Purification and characterization of a sodium-stimulated β-galactosidase from Bifidobacterium longum CCRC 15708

Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg⁻¹ protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C, respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K _m and V _max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na⁺ and K⁺ stimulated the enzyme up to 10-fold, while Fe³⁺, Fe²⁺, Co²⁺, Cu²⁺, Ca²⁺, Zn²⁺, Mn²⁺ and Mg²⁺ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on the enzyme activity for ONPG is probably a case of competitive inhibition. A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects, particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources. Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application.     

Inhibition of CCl4-induced liver fibrosis by Piper longum Linn.?

This study was carried out to evaluate the antifibrotic effect of ethanol extract of the fruits of Indian herb Piper longum Linn. Liver fibrosis was induced in rats by CCl(4) administration. The extent of liver fibrosis was assessed by measuring the level of liver hydroxy proline (HP) and serum enzyme levels. Following CCl(4) administration HP was significantly increased and serum enzyme levels were elevated. Treatment with the ethanol extract of Piper longum Linn. reduced the HP and also the serum enzymes. The liver weight that increased following CCl(4) administration due to the deposition of collagen was reduced by the ethanol extract. Hence, it is concluded that this extract inhibits liver fibrosis induced by CCl(4).     

Die Aminosäuresequenz des Threonin und Serin enthaltenden Mureins von Bifidobacterium longum Reuter

Zusammenfassung Von 18 Stämmen von Bifidobacterium longum Reuter und einem Stamm von B. lactentis Reuter wurden die Zellwände in üblicher Weise hergestellt. Sie enthielten Mur, GlcNH₂, d-Glu, Ala, l-Orn (l-Lys), Thr, Ser in einem Molverhältnis von rund 1:1:1:4:1:1:1. Die Molverhältnisse änderten sich nicht, wenn die Zellwände mit Trichloressigsäure oder heißem Formamid extrahiert wurden. In einigen Stämmen trat mehr als 1 Mol Glutaminsäure pro Mol Diaminosäure auf. Die zusätzliche Glutaminsäure hatte die l-Konfiguration. Sie war kein Bestandteil des Mureins, sondern einer lysozymunempfindlichen, unbekannten Zellwandkomponente, vermutlich einer Polyglutaminsäure. l-Ornithin war in den meisten Stämmen die dominierende Diaminosäure, während l-Lysin nur mit einem Anteil von 10–20% vertreten war. In 2 Stämmen war l-Lysin dominierend (90%). Die Aminosäuresequenz wurde durch die Analyse der Oligopeptide aus Partialhydrolysaten bestimmt. Die an Mureinsäure gebundenen Peptiduntereinheiten hatten die auch von anderen Mureinen bekannte Sequenz: l-Ala-d-Glu-l-Orn (oder l-Lys)-d-Ala. Glutaminsäure ist wahrscheinlich amidiert, wie aus dem Auftreten von rund 1 Mol NH₃ im Hydrolysat der Zellwände zu schließen ist. Die Interpeptidbrücke besteht aus dem Peptid l-Ala-Thr-l-Ala-l-Ser. Sie ist mit dem C-terminalen Serin an die -Aminogruppe der Diaminosäure der Peptiduntereinheit gebunden. Die Quervernetzung erfolgt zwischen dem N-terminalen Alanin der Interpeptidbrücke zum C-terminalen d-Alanin einer Peptiduntereinheit. Da 4% des gesamten Alanins und 3% der -Aminogruppe des Ornithins dinitrophenylierbar sind, ist anzunehmen, daß die Quervernetzung nur zu etwa 80% verwirklicht ist.

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The amino acid sequence of the threonine and serine containing murein of Bifidobacterium longum reuter

Summary Cell walls of 18 strains of Bifidobacterium longum Reuter and one strain of B. lactentis Reuter were prepared in the usual way. They contained Mur, GlcNH₂, d-Glu, Ala, l-Orn (l-Lys), Thr, Ser in a molar ratio of about 1:1:1:4:1:1:1. The ratio was not changed when the cell walls were extracted by trichloroacetic acid or hot formamide. In some strains more than 1 mole glutamic acid per mole of diamino acid was present. The additional glutamic acid was of the l-rather than the d-form. It was not a constituent of the murein, but of an unknown lysozyme insensitive cell wall component, probably a polyglutamic acid. l-Ornithine was the dominating diamino acid in most strains but l-lysine was also present in a portion of 10 to 20%. In 2 strains l-lysine was dominating (90%).The amino acid sequence was determined by analysing the oligopeptides arising during partial acid hydrolysis. It was shown that the peptide subunits attached to the muramic acid are the same as those of other mureins: l-Ala-d-Glu-l-Orn (or l-Lys)-d-Ala. glutamic acid is probably amidated, since about 1 mole of NH₃ is released by acid hydrolysis of the cell walls. The interpeptide bridge consists of the peptide l-Ala-Thr-l-Ala-l-Ser which is bound by its C-terminal serine to the -amino group of the diamino acid of one peptide subunit and by its N-terminal l-alanine to the C-terminal d-alanine of another peptide subunit. About 4% of the total alanine and 3% of the -amino groups of ornithine of the cell wall can be dinitrophenylated. This indicates that about 20% of the peptide subunits are not crosslinked.

     

Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides

Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.     

Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose

We solved the 1.8 ? crystal structure of β-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of β-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The active site is located in the centre of the β-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 β-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 ? crystal structure of the β-fructofuranosidase complex with β-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.     

Cloning and characterization of α-L-arabinofuranosidase and bifunctional α-L-arabinopyranosidase/β-D-galactopyranosidase from Bifidobacterium longum H-1

Aims: This study focused on the cloning, expression and characterization of recombinant α‐l ‐arabinosidases from Bifidobacterium longum H‐1. Methods and Results: α‐l ‐Arabinofuranosidase (AfuB‐H1) and bifunctional α‐l ‐arabinopyranosidase/β‐d ‐galactosidase (Apy‐H1) from B. longum H‐1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB‐H1 (rAfuB‐H1) was purified by single‐step Ni²⁺‐affinity column chromatography, whereas recombinant Apy‐H1 (rApy‐H1) was purified by serial Q‐HP and Ni²⁺‐affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB‐H1 hydrolysed p‐nitrophenyl‐α‐l ‐arabinofuranoside (pNP‐αL‐Af) and ginsenoside Rc, but did not hydrolyse p‐nitrophenyl‐α‐l ‐arabinopyranoside (pNP‐αL‐Ap). On the other hand, rApy‐H1 hydrolysed pNP‐αL‐Ap, p‐nitrophenyl‐β‐d ‐galactopyranoside (pNP‐βD‐Ga) and ginsenoside Rb2. Conclusions: Ginsenoside‐metabolizing bifidobacterial rAfuB‐H1 and rApy‐H1 were successfully cloned, expressed, and characterized. rAfuB‐H1 specifically recognized the α‐l ‐arabinofuranoside, whereas rApy‐H1 had dual functions, that is, it could hydrolyse both β‐d ‐galactopyranoside and α‐l ‐arabinopyranoside. Significance and Impact of the Study: These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified α‐l ‐arabinosidases from Bifidobacterium breve K‐110 and Clostridium cellulovorans.     

Characterization of a novel β-L-Arabinofuranosidase in Bifidobacterium longum: functional elucidation of A DUF1680 family member

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.     

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum: FUNCTIONAL ELUCIDATION OF A DUF1680 PROTEIN FAMILY MEMBER*

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. Previously, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-338 is a critical residue for catalytic activity. This study provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the glycoside hydrolase family 127.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

Development of a double-crossover markerless gene deletion system in Bifidobacterium longum: functional analysis of the α-galactosidase gene for raffinose assimilation

Functional analysis of Bifidobacterium genes is essential for understanding host-Bifidobacterium interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using the aga gene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional α-galactosidase enzyme in B. longum 105-A.