A fragment of the ß-glucosidase gene from the rumen fungus Neocallimastix patriciarum J11 encodes a recombinant protein that exhibits activities in ß-glucosidase and ß-glucanase.

Lignocellulose, the most abundant organic waste on Earth, is of economic value because it can be converted into biofuels like ethanol by enzymes such as ß-glucosidase. This study involved cloning a ß-glucosidase gene named JBG from the rumen fungus Neocallimastix patriciarum J11. When expressed recombinantly in Escherichia coli, the rJBG enzyme exhibited significant activity, hydrolyzing 4-nitrophenyl-ß-d-glucopyranoside and cellobiose to release glucose. Surprisingly, the rJBG enzyme also showed hydrolytic activity against ß-glucan, breaking it down into glucose, indicating that the rJBG enzyme possesses both ß-glucosidase and ß-glucanase activities, a characteristic rarely found in ß-glucosidases. When the JBG gene was expressed in Saccharomyces cerevisiae and the transformants were inoculated into a medium containing ß-glucan as the sole carbon source, the ethanol concentration in the culture medium increased from 0.17 g/L on the first day to 0.77 g/L on the third day, reaching 1.3 g/L on the fifth day, whereas no ethanol was detected in the yeast transformants containing the recombinant plasmid pYES-Sur under the same conditions. These results demonstrate that yeast transformants carrying the JBG gene can directly saccharify ß-glucan and ferment it to produce ethanol. This gene, with its dual ß-glucosidase and ß-glucanase activities, simplifies and reduces the cost of the typical process of converting lignocellulose into bioethanol using enzymes and yeast.

Exopolysaccharides of Bacillus amyloliquefaciens Amy-1 Mitigate Inflammation by Inhibiting ERK1/2 and NF-κB Pathways and Activating p38/Nrf2 Pathway.

Bacillus amyloliquefaciens is a probiotic for animals. Evidence suggests that diets supplemented with B. amyloliquefaciens can reduce inflammation; however, the underlying mechanism is unclear and requires further exploration. The exopolysaccharides of B. amyloliquefaciens amy-1 displayed hypoglycemic activity previously, suggesting that they are bioactive molecules. In addition, they counteracted the effect of lipopolysaccharide (LPS) on inducing cellular insulin resistance in exploratory tests. Therefore, this study aimed to explore the anti-inflammatory effect and molecular mechanisms of the exopolysaccharide preparation of amy-1 (EPS). Consequently, EPS reduced the expression of proinflammatory factors, the phagocytic activity and oxidative stress of LPS-stimulated THP-1 cells. In animal tests, EPS effectively ameliorated ear inflammation of mice. These data suggested that EPS possess anti-inflammatory activity. A mechanism study revealed that EPS inhibited the nuclear factor-κB pathway, activated the mitogen-activated protein kinase (MAPK) p38, and prohibited the extracellular signal-regulated kinase 1/2, but had no effect on the c-Jun-N-terminal kinase 2 (JNK). EPS also activated the anti-oxidative nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Evidence suggested that p38, but not JNK, was involved in activating the Nrf2 pathway. Together, these mechanisms reduced the severity of inflammation. These findings support the proposal that exopolysaccharides may play important roles in the anti-inflammatory functions of probiotics.

Cytotoxic and Anti-Inflammatory Triterpenoids in the Vines and Leaves of Momordica charantia.

The vines and leaves of Momordica charantia L. are used as herbal medicines to treat inflammation-related disorders. However, their safety profile remains uncharacterized, and the constituents in their extracts that exert anti-inflammatory and adverse effects remain unclear. This study isolated the characteristic cucurbitane-type triterpenoid species in the vines and leaves of M. charantia L. and analyzed their cytotoxicity, anti-inflammatory effects, and underlying mechanisms. Four structurally related triterpenoids-momordicines I, II, IV, and (23E) 3ß,7ß,25-trihydroxycucurbita-5,23-dien-19-al (TCD)-were isolated from the triterpenoid-rich fractions of extracts from the vines and leaves of M. charantia. Momordicine I was cytotoxic on normal cells, momordicine II exerted milder cytotoxicity, and momordicine IV and TCD had no obvious adverse effects on cell growth. TCD had anti-inflammatory activity both in vivo and in vitro. In lipopolysaccharide-stimulated RAW 264.7 cells, TCD inhibited the inhibitor kappa B kinase/nuclear factor-κB pathway and enhanced the expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, and glutamate-cysteine ligase modifier subunit through the extracellular signal-regulated kinase1/2 and p38. Thus, the vines and leaves of M. charantia should be used with caution. An extraction protocol that can enrich TCD but remove momordicine I would likely enhance the safety of the extract.

Bitter Melon Extract Yields Multiple Effects on Intestinal Epithelial Cells and Likely Contributes to Anti-diabetic Functions.

The intestines have been recognized as important tissues for metabolic regulation, including glycemic control, but their vital role in promoting the anti-diabetic effects of bitter melon, the fruit of Momordica charantia L, has seldom been characterized, nor acknowledged. Evidence suggests that bitter melon constituents can have substantial interactions with the intestinal epithelial cells before circulating to other tissues. We therefore characterized the effects of bitter melon extract (BME) on intestinal epithelial cells. BME was found to contain substantial amounts of carbohydrates, proteins, and triterpenoids. TNF-α induced insulin resistance in an enterocyte cell line of IEC-18 cells, and BME promoted glucose utilization of the insulin-resistant cells. Further analysis suggested that the increased glucose consumption was a result of the combined effects of insulin sensitizing and insulin substitution functions of BME. The functions of insulin substitution were likely generated due to the activation of AMP-activated protein kinase. Meanwhile, BME acted as a glucagon-like peptide 1 (GLP-1) secretagogue on enteroendocrine cells, which may be mediated by the activation of bitter-taste receptors. Therefore, BME possesses insulin sensitizing, insulin substitution, and GLP-1 secretagogue functions upon intestinal cells. These effects of BME on intestinal cells likely play a significant part in the anti-diabetic action of bitter melon.

The antimicrobial activities of phenylbutyrates against Helicobacter pylori.

Three phenyl derivatives of butyrate, 2-phenylbutyrate (2-PB), 3-phenylbutyrate (3-PB) and 4-phenylbutyrate (4-PB), were evaluated in terms of their antibacterial and cytotoxic activities. Our results indicated that PBs demonstrated specific inhibitory activity against Helicobacter pylori and Escherichia coli but did not influence the growth of Bifidobacterium bifidium and Lactobacillus reuteri. PBs also exhibited synergistic effects on H. pylori ATCC 43504 especially at pH 5.5. In the protein expression profiles in H. pylori treated by phenylbutyrates, we also found that three protein spots identified as oxidative stress-related proteins were significantly up-regulated, confirming the response of H. pylori when exposed to PBs. Due to their antibacterial activities and low or slight cytotoxicities, PBs are potential candidates for the treatment of H. pylori infection. This is the first study to discover the antibiotic effects of 2-PB, 3-PB and 4-PB (Buphenyl).

Biological activity of oleanane triterpene derivatives obtained by chemical derivatization.

Nine new derivatives of oleanane triterpenoids isolated from Fatsia polycarpa Hayata were synthesized through chemical transformations. Acetylation was effected by reaction with acetic anhydride in pyridine to afford compounds 1-5, while compound 6 was obtained using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in CH2Cl2. The others derivatives 7-9 were obtained in reactions of the corresponding triterpenoids with EDC·HCl, 4-N,N-dimethylaminopyridine hydrochloride and 4-N,N-dimethylaminopyridine in CH2Cl2. The structures of 1-9 were elucidated from extensive spectroscopic and HRESIMS data, while the structure of 9 was further confirmed by X-ray diffraction analysis. The cytotoxic, anti-hepatitis B virus (HBV), antibacterial, hypoglycaemic and Wnt signaling activities of these derivatives were evaluated in vitro.

Bacillus amyloliquefaciens exopolysaccharide preparation induces glucagon-like peptide 1 secretion through the activation of bitter taste receptors.

The exopolysaccharide preparation of Bacillus amyloliquefaciens amy-1 (EPS) regulates glycemic levels and promotes glucagon-like peptide 1 (GLP-1) secretion in vivo and in vitro. This study aimed to identify the molecular mechanism underlying EPS-induced GLP-1 secretion. HEK293T cells stably expressing human Gα-gustducin were used as a heterologous system for expressing the genes of human bitter taste receptor (T2R) 10, 14, 30, 38 (PAV), 38 (AVI), 43, and 46, which were expressed as recombinant proteins with an N-terminal tag composed of a Lucy peptide and a human somatostatin receptor subtype 3 fragment for membrane targeting and a C-terminal red fluorescent protein for expression monitoring. EPS induced a dose-dependent calcium response from the human NCI-H716 enteroendocrine cell line revealed by fluorescent calcium imaging, but inhibitors of the G protein-coupled receptor pathway suppressed the response. EPS activated heterologously expressed T2R14 and T2R38 (PAV). shRNAs of T2R14 effectively inhibited EPS-induced calcium response and GLP-1 secretion in NCI-H716 cells, suggesting the involvement of T2R14 in these effects. The involvement of T2R38 was not characterized because NCI-H716 cells express T2R38 (AVI). In conclusion, the activation of T2Rs mediates EPS-induced GLP-1 secretion from enteroendocrine cells, and T2R14 is a critical target activated by EPS in these cells.

Cucurbitane triterpenoids from Momordica charantia and their cytoprotective activity in tert-butyl hydroperoxide-induced hepatotoxicity of HepG2 cells.

Two novel pentanorcucurbitane triterpenes, 22-hydroxy-23,24,25,26,27-pentanorcucurbit-5-en-3-one (1) and 3,7-dioxo-23,24,25,26,27-pentanorcucurbit-5-en-22-oic acid (2) together with a new trinorcucurbitane triterpene, 25,26,27-trinorcucurbit-5-ene-3,7,23-trione (3) were isolated from the methyl alcohol extract of the stems of Momordica charantia. The structures of the new compounds were elucidated by spectroscopic methods. Compounds 2 and 3 showed potent cytoprotective activity in tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity of HepG2 cells.

Octanorcucurbitane triterpenoids protect against tert-butyl hydroperoxide-induced hepatotoxicity from the stems of Momordica charantia.

Four novel octanorcucurbitane triterpenes, octanorcucurbitacins A-D (1-4), together with one known octanorcucurbitane triterpene, kuguacin M (5), were isolated from the methyl alcohol extract of the stems of Momordica charantia. Their structures were elucidated on the basis of extensive spectroscopic analyses. Compound 3 inhibited tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity against HepG2 cells.

Exopolysaccharides of Bacillus amyloliquefaciens modulate glycemic level in mice and promote glucose uptake of cells through the activation of Akt.

Bacillus amyloliquefaciens is a probiotic for animals. A strain of B. amyloliquefaciens designated amy-1 was isolated from soil, and the exopolysaccharides (EPSs) of the strain were characterized in terms of their effect on glycemic control. The EPSs were composed of mannose, glucose, and galactose, with the major components being polymers larger than 1000 kDa as revealed by size-exclusion high-performance liquid chromatography. The EPSs reduced the elevation of blood glucose in mice on oral glucose tolerance tests. The hypoglycemic effect was still apparent when glucose was administered through intraperitoneal injection. Further investigation revealed that the EPSs stimulated glucagon-like peptide 1 (GLP-1) secretion from enteroendocrine cells in vitro and increased plasma GLP-1 level in vivo. Moreover, the EPSs promoted the glucose consumption of a liver cell line and an intestinal epithelial cell line. Therefore, the interaction between EPSs and intestinal tissues at least partially contributed to their hypoglycemic effect. The enhanced glucose uptake of cells was likely mediated by the activation of phosphatidylinositol-3-kinase and Akt and was independent of insulin receptor substrate and AMP-activated protein kinase. These findings suggest that EPSs likely involve in the hypoglycemic functions of probiotics and are potential new agents for glycemic control.

The identification, purification, and characterization of STXF10 expressed in Streptomyces thermonitrificans NTU-88.

Multiple xylanolytic enzymes of Streptomyces thermonitrificans NTU-88 were induced by oat-spelt xylan and separated by two-dimensional polyacrylamide and zymogram gels. Nineteen clear spots differed in pI and molecular weight values were found on the zymogram, and only spot one was seen on the corresponding silver-stained gel. These results revealed that multiple xylanases were secreted when S. thermonitrificans NTU-88 was induced and the spot (STXF10), identified as being a glycosyl hydrolase family 10 xylanase, was the predominant one among xylanases. STXF10 showed a tolerance for high temperatures and broad pH ranges and high affinity and hydrolysis efficiency for xylans. Furthermore, it also featured the minor ability to degrade different lignocellulosic substrates. Although S. thermonitrificans NTU-88 possesses multiple xylanases, our results suggest that the major form of xylanase might be selectively and specifically induced depending on the type of substrate to which the microorganism is exposed.

Cucurbitane-type triterpenoids from the stems of momordica charantia.

Four new cucurbitane-type triterpenes, cucurbita-5,23(E)-diene-3beta,7beta,25-triol (1), 3beta-acetoxy-7beta-methoxycucurbita-5,23(E)-dien-25-ol (2), cucurbita-5(10),6,23(E)-triene-3beta,25-diol (5), and cucurbita-5,24-diene-3,7,23-trione (6), together with four known triterpenes, 3beta,25-dihydroxy-7beta-methoxycucurbita-5,23(E)-diene (3), 3beta-hydroxy-7beta,25-dimethoxycucurbita-5,23(E)-diene (4), 3beta,7beta,25-trihydroxycucurbita-5,23(E)-dien-19-al (7), and 25-methoxy-3beta,7beta-dihydroxycucurbita-5,23(E)-dien-19-al (8), were isolated from the methyl alcohol extract of the stems of Momordica charantia. The structures of the new compounds were elucidated by spectroscopic methods.

Cloning, characterization and phylogenetic relationships of stxI, a endoxylanase-encoding gene from Streptomyces thermonitrificans NTU-88.

A thermostable xylanase gene (stxI) obtained from Streptomyces thermonitrificans NTU-88 on domain analysis revealed an N-terminal catalytic domain featuring homology to a known xylanase within the glycoside hydrolase family 11. Recombinant STXI retained more than 60% of its activity following its incubation for at 60 degrees C for 24h. These characteristics were close to thermophile and mesophile Streptomyces strains. The main hydrolysis products of xylan degraded by STXI included large xylooligosaccharide fragments. These results indicated that STXI was a typical endoxylanase. As regards the phylogenetic relationships of GH11, STXI and the other xylanase deriving from Streptomyces were included in a subgroup of the aerobic bacterial group. This result implied that the evolutionary relationships between the various xylanases deriving from Streptomyces strains were convergent.

Caecomyces sympodialis sp. nov., a new rumen fungus isolated from Bos indicus.

A new anaerobic rumen fungus was isolated from the rumen fluid of a yellow cow (Bos indicus). This fungus appears to be a previously undescribed species of the genus Caecomyces, it possessing uniflagellate zoospores, a spherical holdfast, tubular sporangiophores and bulbous rhizoids. This new fungus also features distinctive multisporangiate thallus sympodially distributed on sporangiophores. The fungus resembles Caecomyces communis and C. equi in that it characterizes bulbous rhizoids and uniflagellate zoospores but differs from C. communis and C. equi in that it possesses multisporangiate and sympodial sporangia. This new fungus and Cyllamyces aberensis both reveal similar morphology during early thallus development in having a spherical holdfast, but they vary from unbranched sporangiophores and additional bulbous rhizoids. In addition, the molecular phylogenetic analyses ITS1 (internal transcribed spacer 1) also conform to the results of the morphological examinations of Caecomyces. For the mentioned reasons, this new species of fungus is described as Caecomyces sympodialis sp. nov. The genera of Neocallimasticaceae and species of Caecomyces are also keyed out.

A highly active beta-glucanase from a new strain of rumen fungus Orpinomyces sp.Y102 exhibits cellobiohydrolase and cellotriohydrolase activities.

A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1-17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to ß-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64µmole/min/mg and a Km of 2.18mg/ml to barley ß-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.

Characterization of two truncated forms of xylanase recombinantly expressed by Lactobacillus reuteri with an introduced rumen fungal xylanase gene.

The xylanase R8 gene (xynR8) from uncultured rumen fungi was cloned and successfully expressed in Lactobacillus reuteri. A xylanase activity of 132.1 U/mL was found in the broth of L. reuteri R8, the transformant containing pNZ3004 vector with xynR8 gene insertion. Two distinct forms of recombinant xylanase with different hydrophobicities and molecular weights were found in the broth after purification. According to the results of Western blotting, only the T7-tag, fused in the N-terminus of XynR8, could be bound to the expressed proteins, which indicated that the C-terminus of XynR8 had been truncated. These results, combined with tryptic digestion and mass spectrometry analyses, allow us to attribute the two xylanase forms to an optional cleavage of C-terminal sequences, and XynR8A, a 13 amino acid residues truncated form, and XynR8B, a 22 amino acid residues truncated form, were the main products in the extracellular fraction of L. reuteri R8. The specific activities of XynR8A and R8B were 1028 and 395 U/mg protein. Both forms of recombinant xylanase displayed a typical endoxylanase activity when they were reacted with xylan, but XynR8A demonstrated a better specific activity, catalytic efficiency and thermostability than XynR8B according to the results of enzyme characterization. These changes in enzyme properties were highly possibly caused by the present of the ß-sheet in the C-terminal undeleted fragment of XynR8A. This study demonstrates that modified forms with different enzyme properties could be produced when a gene was recombinantly expressed by a L. reuteri transformant.

Anti-Inflammatory Effects and Mechanisms of Fatsia polycarpa Hayata and Its Constituents.

Fatsia polycarpa, a plant endemic to Taiwan, is an herbal medicine known for treating several inflammation-related diseases, but its biological function needs scientific support. Thus, the anti-inflammatory effects and mechanisms of the methanolic crude extract (MCE) of F. polycarpa and its feature constituents, that is, brassicasterol (a phytosterol), triterpenoids 3 α -hydroxyolean-11,13(18)-dien-28-oic acid (HODA), 3 α -hydroxyolean-11-en-28,13 ß -olide (HOEO), fatsicarpain D, and fatsicarpain F, were investigated. MCE and HOEO, but not brassicasterol, dose-dependently inhibited lipopolysaccharide- (LPS-)induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophage line, whereas HODA, fatsicarpain D and fatsicarpain F were toxic to RAW cells. Additionally, MCE and HOEO suppressed LPS-induced production of nitric oxide, prostaglandin E2, and interleukin-1 ß and interfered with LPS-promoted activation of the inhibitor kappa B kinase (IKK)/nuclear factor- κ B (NF- κ B) pathway, and that of the mitogen-activated protein kinases (MAPKs) extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. In animal tests, MCE and HOEO effectively ameliorated 12-O-tetradecanoylphorobol-13 acetate- (TPA-)induced ear edema of mice. Thus, MCE of F. polycarpa exhibited an obvious anti-inflammatory activity in vivo and in vitro that likely involved the inhibition of the IKK/NF- κ B pathway and the MAPKs, which may be attributed by triterpenoids such as HOEO.

Norcucurbitane triterpenoids from the fruits of Momordica charantia var. abbreviata.

Two new 27-norcucurbitane triterpenoids, 27-nor-3beta-hydroxy-7beta-methoxycucurbita-5,23(E)-dien-25-one (1) and 27-nor-3beta-hydroxy-5beta,19-epoxycucurbita-6,23(E)-dien-25-one (2), together with two known cucurbitane triterpenes, 23(E)-7beta-methoxycucurbita-5,23,25-trien-3beta-ol (3) and 5beta,19-epoxy-25-methoxycucurbita-6,23(E)-dien-3beta-ol (4), were isolated from the fruits of Momordica charantia var. abbreviata. Their structures were determined by analysis of spectroscopic data and comparison with the data of known analogues.

EMCD, a hypoglycemic triterpene isolated from Momordica charantia wild variant, attenuates TNF-α-induced inflammation in FL83B cells in an AMP-activated protein kinase-independent manner.

Insulin resistance is a causative factor for type 2 diabetes, whereas the development of insulin resistance is closely related to chronic inflammation induced by factors such as tumor necrosis factor-α (TNF-α). Momordica charantia, also known as bitter melon, has been used as an herbal medicine and reported to ameliorate inflammation and hyperglycemia. Previously, a triterpene 5ß,19-epoxy-25-methoxy-cucurbita-6,23-diene-3ß,19-diol (EMCD), purified from M. charantia L. wild variant WB24, was found to activate AMP-activated protein kinase (AMPK) and have a hypoglycaemic effect in TNF-α-treated FL83B cells. AMPK has been a target for developing anti-diabetic medicine and suggested to play a role in anti-inflammation. The current study aims to investigate if EMCD might repress TNF-α-induced inflammation via AMPK. TNF-α-induced inflammation in FL83B cells was characterized using Western blotting and reverse transcriptase-polymerase chain reaction. Consequently, the expression of inflammatory markers including inducible nitric oxide synthase (iNOS), the p65 subunit of nuclear factor-κB (NF-κB), protein-tyrosine phosphatase-1B, TNF-α and interleukin-1ß were significantly elevated by TNF-α in the cell, and EMCD obviously suppressed the TNF-α-induced expression of these markers. When the effect of EMCD was tested simultaneously with epigallocatechin-3-gallate (EGCG), a catechin from green tea reported to be anti-inflammatory, EMCD showed a more obvious anti-inflammatory activity than EGCG did. Investigation of the underlying mechanism suggested that EMCD inhibited the activation of the IκB kinase (IKK) complex and the NF-κB pathway, and the effect was likely independent of AMPK. Collectively, the multiple functions of EMCD suggest it to be a potential agent in treating diabetic complications and other inflammation-related disorders.

Structural modeling and further improvement in pH stability and activity of a highly-active xylanase from an uncultured rumen fungus.

Rumen fungi are a rich source of enzymes degrading lignocelluloses. XynR8 is a glycosyl hydrolase family 11 xylanase previously cloned from unpurified rumen fungal cultures. Phylogenetic analysis suggested that xynR8 was obtained from a Neocallimastix species. Recombinant XynR8 expressed in Escherichia coli was highly active and stable between pH 3.0 and 11.0, and displayed a V(max) of 66,672µmolmin(-1)mg(-1), a k(cat) of 38,975s(-1), and a K(m) of 11.20mg/mL towards soluble oat spelt xylan. Based on molecular modeling, residues N41 and N58, important in stabilizing two loops and the structure of XynR8, were mutated to D. Both mutant enzymes showed higher tolerance to pH 2.0. The V(max), k(cat) and K(m) of the N41D and N58D mutant enzymes were 79,645µmolmin(-1)mg(-1), 46,493s(-1), 29.29mg/mL, and 96,689µmolmin(-1)mg(-1), 56,503s(-1), and 21.24mg/mL, respectively. Thus, they are good candidates for application, including biofuel production.