Synthesis of 2'-fucosyllactose from apple pomace-derived xyloglucan oligosaccharides by an α-L-fucosidase from Pedobacter sp. CAU209.

2'-Fucosyllactose (2'-FL) is known for its ability to provide various health benefits to infants, such as gut maturation, pathogen resistance, improved immunity, and nervous system development. However, the production of 2'-FL using α-L-fucosidases is hindered by the lack of low-cost natural fucosyl donors and high-efficiency α-L-fucosidases. In this work, a recombinant xyloglucanase from Rhizomucor miehei (RmXEG12A) was applied to produce xyloglucan-oligosaccharide (XyG-oligos) from apple pomace. Then, an α-L-fucosidase gene (PbFucB) was screened from the genomic DNA of Pedobacter sp. CAU209 and expressed in Escherichia coli. The capability of purified PbFucB to catalyze XyG-oligos and lactose to synthesize 2'-FL was further evaluated. The deduced amino acid sequence of PbFucB shared the highest identity (38.4%) with that of other reported α-L-fucosidases. PbFucB showed the highest activity at pH 5.5 and 35 °C. It catalyzed the hydrolysis of 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc, 20.3 U mg-1), 2'-FL (8.06 U mg-1), and XyG-oligos (0.43 U mg-1). Furthermore, PbFucB demonstrated a high enzymatic conversion rate in 2'-FL synthesis with pNP-Fuc or apple pomace-derived XyG-oligos as donors and lactose as acceptor. Under the optimized conditions, PbFucB converted 50% of pNP-Fuc or 31% of the L-fucosyl residue in XyG-oligos into 2'-FL. This work elucidated an α-L-fucosidase that mediates the fucosylation of lactose and provided an efficient enzymatic strategy to synthesize 2'-FL either from artificial pNP-Fuc or natural apple pomace-derived XyG-oligos. KEY POINTS: • Xyloglucan-oligosaccharide (XyG-oligos) was produced from apple pomace by a xyloglucanase from Rhizomucor miehei. • An α-L-fucosidase (PbFucB) from Pedobacter sp. CAU209 shared the highest identity (38.4%) with reported α-L-fucosidases. •PbFucB synthesized 2'-FL using apple pomace-derived XyG-oligos and lactose with a conversion ratio of 31%.

Efficient sequential synthesis of lacto-N-triose II and lacto-N-neotetraose by a novel ß-N-acetylhexosaminidase from Tyzzerella nexilis.

ß-N-acetylhexosaminidases have attracted much attention in recent years due to their potential application in oligosaccharide production, in particular lacto-N-triose II (LNT2) and lacto-N-neotetraose (LNnT) synthesis, which can be further used as backbone precursors for human milk oligosaccharides. A novel ß-N-acetylhexosaminidase gene from Tyzzerella nexilis (TnHex189) was heterologously expressed in Bacillus subtilis. The highest ß-N-acetylhexosaminidase activity of 14.5 U mL-1 was obtained in a 5-L fermentor by fed-batch fermentation for 27 h. TnHex189 was optimally active at pH 5.0 and 45 °C. It efficiently synthesized LNT2 with a conversion ratio of 57.2% (4.7 g L-1). The synthesized LNT2 was further converted to LNnT by a reported ß-galactosidase (BgaD-D) in 8 h, with a conversion ratio of 17.3% (6.1 g L-1). These unique synthesis activities may make this enzyme a good candidate for the food industry.

Geniposide and Gentiopicroside Suppress Hepatic Gluconeogenesis via Regulation of AKT-FOXO1 Pathway.

BACKGROUND: Hepatic gluconeogenesis plays an important role in regulating fasting plasma glucose levels and is a target of anti-diabetic drugs. Several kinds of iridoid glucosides exhibit hypoglycemic effect, whereas the mechanism was not clear. AIM OF THE STUDY: In this study, the effects of geniposide and gentiopicroside, two natural iridoid glucosides, on hepatic gluconeogenesis were investigated. METHODS: Glucose uptake assay, MTT assay, q-PCR, luciferase assay and western blot assay were performed to investigate the pharmacological effect of geniposide and gentiopicroside on human liver cell line L02. Thereby the fast blood glucose and intraperitoneal glucose tolerance were measured in high fat diet induced hyperglycemic mice after geniposide or gentiopicroside administration. RESULTS: The results showed that geniposide and gentiopicroside inhibited the transcription of G6PC and PEPCK in L02 cells and in mice. Additional experimental data indicated that these two compounds were able to inhibit the transcriptional activity of FOXO1 by inducing phosphorylation of AKT at Ser473. Furthermore, we found that these two compounds alleviated high fat diet induced hyperglycemia in mice. CONCLUSIONS: Geniposide and gentiopicroside might reduce blood glucose and suppress hepatic gluconeogenesis by regulating the AKT-FOXO1 pathway, and the potential use of these two iridoid glucosides as anti-diabetic agents merits further in-depth exploration.

Antimicrobial mechanism of theaflavins: They target 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 µM) and theaflavin-3,3'-digallate exhibiting the highest (IC50 14.9 µM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments.

A specific process to purify 2-methyl-D-erythritol-4-phosphate enzymatically converted from D-glyceraldehyde-3-phosphate and pyruvate.

A one-pot enzymatic cascade was established to synthesize MEP, one of the key intermediates in the MEP terpenoid biosynthetic pathway. D-GAP and sodium pyruvate were converted to MEP in a reaction catalyzed by DXP synthase and DXP reductoisomerase (DXR) in the presence of the coenzymes ThPP, NADPH, and Mg2+. The product was then isolated by using a specific two-step purification process and MEP was obtained in a yield of nearly 60% and high purity. Importantly, MEP prepared by this way was totally free from contamination by minor amounts of DXP that was not completely convertible by DXR.

Biosynthesis of the iridoid glucoside, lamalbid, in Lamium barbatum.

The biosynthesis of the iridoid glucoside lamalbid in Lamium barbatum, a plant species in the Lamiaceae, was investigated by administrating (13)C-labeled intermediates of MVA and MEP pathways, respectively. The results demonstrated that [3,4,5-(13)C(3)]1-deoxy-D-xylulose 5-phosphate could be incorporated into lamalbid, whereas the incorporation of [2-(13)C(1)]mevalonolactone was not observed. Based on the (13)C labeling pattern of lamalbid and the incorporation data, we deduce that the iridoid glucoside in L. barbatum is biosynthesized through the MEP pathway, whereas the classic MVA pathway is not utilized.

Study of antiepileptic effect of extracts from Acorus tatarinowii Schott.

PURPOSE: To study the antiepileptic properties of extracts from rhizomes of Acorus tatarinowii Schott (ATS). METHODS: The decoction and volatile oil were extracted from rhizomes of ATS by traditional decocting and supercritical CO(2) fluid extraction (SFE-CO(2)) methods. Maximal electroshock (MES), pentylenetetrazol (PTZ) maximal seizure, and prolonged PTZ kindling models were used to test their anticonvulsive properties. The gamma-aminobutyric acid (GABA) immunohistochemical reaction (IR) was used to study GABAergic neuron changes in the PTZ kindling model and the effects of treatment. RESULTS: Both decoction (dose; 10-20 g/kg) and volatile oil (1.25 g/kg) of ATS decreased the convulsive rate significantly in the MES model. Decoction of ATS was shown to be effective in the PTZ model with both decreased convulsive and mortality rates. The volatile oil of ATS failed to prevent seizures in the dose range tested, although prolonged seizure latency and decreased mortality were found at a dose of 1.25 g/kg. In the PTZ kindling model, GABA-IR neurons decreased obviously compared with the normal group. In the groups treated with the decoction and volatile oil, the seizure intensity decreased significantly after treatment. Increased GABA-IR neurons also were found when compared with PTZ kindling controls. Morphologic observation also showed that GABA-IR neuron damage was less severe in the drug-treated groups. CONCLUSIONS: Both decoction and volatile oil extracted from the rhizome of Acorus tatarinowii Schott have anticonvulsive effects. The volatile oil is shown to be less effective for PTZ-induced convulsions. Both extracts can prevent convulsions as well as convulsion-related GABAergic neuron damage in the brain in the prolonged PTZ kindling model.