Aerobic bioconversion of C-glycoside mangiferin into its aglycone norathyriol by an isolated mouse intestinal bacterium.

Norathyriol is an aglycone of a xanthonoid C-glycoside mangiferin that possesses different bioactive properties useful for humans compared to mangiferin. Mangiferin is more readily available in nature than norathyriol; thus, efficient mangiferin conversion into norathyriol is desirable. There are a few reports regarding mangiferin C-deglycosylation because of the C-C bond resistance toward acid, alkaline, and enzyme hydrolysis. In this study, we isolated a mangiferin-deglycosylating bacterium strain KM7-1 from the mouse intestine. 16S rDNA sequencing indicated that KM7-1 belongs to the Bacillus genus. Compared to the taxonomically similar bacteria, the growth characteristic of facultative anaerobic and thermophilic resembled, yet only Bacillus sp. KM7-1 was able to convert mangiferin into norathyriol. Resting cells of Bacillus sp. KM7-1 obtained from aerobic cultivation at 50 °C showed high norathyriol formation from 1 m m of mangiferin. Norathyriol formation can be conducted either under aerobic or anaerobic conditions, and the reaction depended on time and bacterial amount.

New analogs of pochonicine, a potent ß-N-acetylglucosaminidase inhibitor from fungus Pochonia suchlasporia var. suchlasporia TAMA 87.

Three novel analogs of pochonicine (1) were isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87, and their structures were elucidated as 7-deoxypochonicine (2), 6-deoxypochonicine (3), and 6,7-dideoxypochonicine (4). These analogs were found to possess the same stereochemistry as pochonicine. Comparison of ß-N-acetylglucosaminidase (GlcNAcase) inhibitory activity between these analogs and pochonicine suggested that the C-6 hydroxy group of pochonicine was essential to its potent GlcNAcase inhibitory activity and that the C-7 hydroxy group also contributed to the activity, but to a lesser extent than the C-6 hydroxy group.

A new asteltoxin analog with insecticidal activity from Pochonia suchlasporia TAMA 87.

A new asteltoxin analog, named asteltoxin H (1), was isolated by the solid-state fermentation of the fungus Pochonia suchlasporia var. suchlasporia TAMA 87. The chemical structure of 1 was deduced by spectroscopic methods, including 1D and 2D NMR, HRESIMS, and UV-Vis analyses. Compound 1 showed insecticidal activity against prepupae of the blowfly, Lucilia sericata, with an LD50 value of 0.94 µg/mg prepupal body weight.

Convenient preparation of an antigenic oligosaccharide from white kidney bean powder: A useful plant oligosaccharide for synthesis of immunoactive glycopolymer.

Plant glycoproteins, especially allergenic glycoproteins such as pollen allergens, often carry antigenic N-glycans with α1-3 fucose and/or ß1-2 xylose residue(s) on the trimannosyl core structure. We previously reported that one of such antigenic free-form N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX) suppressed IL-4 production from Th2 cells of pollinosis patients. For the molecular-level analysis of this immunoactivity, an effective and convenient procedure for large scale preparation of the immunoactive free-form N-glycan and a synthesis of glycopolymers bearing multivalent M3FX has been required. During the preparation of prebiotic oligosaccharides from several edible beans, we found that the free-form M3FX accumulates in relatively large amounts in white kidney beans. In this report, we describe a new procedure for preparation of M3FX from white kidney bean powders by a combination of ion-exchange method, gel-filtration, and hydrophilic partitioning. The high-purity of M3FX prepared by this procedure was confirmed by MS-analysis and 1H-NMR, suggesting that the free-form M3FX can be used for the synthesis of neoglycopolymer. Using this new procedure, the immunoactive oligosaccharide can be prepared without the chemical method such as hydrazinolysis and other purification steps required to exclude other type of N-glycans.

Use of wood vinegar to enhance 5-aminolevulinic acid production by selected Rhodopseudomonas palustris in rubber sheet wastewater for agricultural use.

This study aimed to produce inexpensive 5-aminolevulinic acid (ALA) in a non-sterile latex rubber sheet wastewater (RSW) by Rhodopseudomonas palustris TN114 and PP803 for the possibility to use in agricultural purposes by investigating the optimum conditions, and applying of wood vinegar (WV) as an economical source of levulinic acid to enhance ALA content. The Box-Behnken Design experiment was conducted under microaerobic-light conditions for 96 h with TN114, PP803 and their mixed culture (1:1) by varying initial pH, inoculum size (% v/v) and initial chemical oxygen demand (COD, mg/L). Results showed that the optimal condition (pH, % inoculum size, COD) of each set to produce extracellular ALA was found at 7.50, 6.00, 2000 for TN114; 7.50, 7.00, 3000 for PP803; and 7.50, 6.00, 4000 for a mixed culture; and each set achieved COD reduction as high as 63%, 71% and 75%, respectively. Addition of the optimal concentration of WV at mid log phase at 0.63% for TN114, and 1.25% for PP803 and the mixed culture significantly increased the ALA content by 3.7-4.2 times (128, 90 and 131 µM, respectively) compared to their controls. ALA production cost could be reduced approximately 31 times with WV on the basis of the amount of levulinic acid used. Effluent containing ALA for using in agriculture could be achieved by treating the RSW with the selected ALA producer R. palustris strains under the optimized condition with a little WV additive.

Unique antimicrobial spectrum of ophiobolin K produced by Aspergillus ustus.

A co-cultivation study of two fungal strains showed that Aspergillus ustus could inhibit Aspergillus repens growth. The bioactive compound responsible for the observed activity was purified and identified as a sesterterpene, ophiobolin K. Ophiobolin K exhibited marked inhibition against both fungi and bacteria, especially A. repens, A. glaucus and gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus.

Characterization of exopolymeric substances from selected Rhodopseudomonas palustris strains and their ability to adsorb sodium ions.

Removal of Na(+) by binding with exopolymeric substances (EPS) from Rhodopseudomonas palustris TN114 and PP803 was investigated. The moderate negative correlation pairs (rp) between remaining Alcian blue and amount of Na(+) adsorbed on EPS from strains TN114 and PP803 were -0.652 and -0.609. Both strains showed positive relationships between the amounts of EPS produced and bacterial growth. EPS from strain PP803 had a higher efficiency in removing Na(+) than the EPS from strain TN114 based on their EC50 values (1.79 and 1.49 mg/mL for TN114 and PP803, respectively). The principal component from EPS of strain PP803 which was responsible for salt removal was purified and it was identified as a polysaccharide (≈18 kDa) mainly composed of galacturonic acid. Overall results suggested that EPS is a key factor that our strains used to bind Na(+) allowing their survival in high NaCl concentrations.

Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes

Background: Rice is globally one of the most important food crops, and NaCl stress is a key factor reducing rice yield. Amelioration of NaCl stress was assessed by determining the growth of rice seedlings treated with culture supernatants containing 5-aminolevulinic acid (ALA) secreted by strains of Rhodopseudomonas palustris (TN114 and PP803) and compared to the effects of synthetic ALA (positive control) and no ALA content (negative control). Results: The relative root growth of rice seedlings was determined under NaCl stress (50 mM NaCl), after 21 d of pretreatment. Pretreatments with 1 μM commercial ALA and 10X diluted culture supernatant of strain TN114 (2.57 μM ALA) gave significantly better growth than 10X diluted PP803 supernatant (2.11 μM ALA). Rice growth measured by dry weight under NaCl stress ordered the pretreatments as: commercial ALA N TN114 N PP803 N negative control. NaCl stress strongly decreased total chlorophyll of the plants that correlated with non-photochemical quenching of fluorescence (NPQ). The salt stress also strongly increased hydrogen peroxide (H2O2) concentration in NaCl-stressed plants. The pretreatments were ordered by reduction in H2O2 content under NaCl stress as: commercial ALA N TN114 N PP803 N negative control. The ALA pretreatments incurred remarkable increases of total chlorophyll and antioxidative activities of catalase (CAT), ascorbate peroxide (APx), glutathione reductase (GR) and superoxide dismutase (SOD); under NaCl stress commercial ALA and TN114 had generally stronger effects than PP803. Conclusions: The strain TN114 has potential as a plant growth stimulating bacterium that might enhance rice growth in saline paddy fields at a lower cost than commercial ALA.

Synthesis of eight stereoisomers of pochonicine: nanomolar inhibition of ß-N-acetylhexosaminidases.

Pochonicine, the first naturally occurring polyhydroxylated pyrrolizidine containing an acetamidomethyl group, which was isolated from Pochonia suchlasporia var. suchlasporia TAMA 87, together with its enantiomer and their C-1 and/or C-3 epimers, have been synthesized from the sugar-derived cyclic nitrones 9D and 9L, respectively. An in-depth NMR study showed that both the (1)H and (13)C NMR spectra of the synthetic pochonicines (1D and 1L) matched very well with those of natural pochonicine in D2O, which unequivocally determined the relative configuration of the natural product as 1D or 1L. In addition, comparison of the optical rotations of the synthetic pochonicines and that of the natural product, but more convincingly their glycosidase inhibition profiles, confirmed the absolute configuration of natural pochonicine as 1R,3S,5R,6R,7S,7aR. Thereby, the structure of natural pochonicine was unequivocally determined as (+)-(1R,3S,5R,6R,7S,7aR)-pochonicine (1D). Glycosidase inhibition experiments showed that natural pochonicine 1D and its epimers 2D, 3D, and 4D all are powerful inhibitors of hexosaminidases (five ß-N-acetylglucosaminidases and two ß-N-acetylgalactosaminidases) while their enantiomers 1L, 2L, 3L, and 4L are much weaker inhibitors of the same enzymes. (-)-3-epi-Pochonicine (2L) was found to be a potent and selective inhibitor of α-l-rhamnosidase. None of the compounds showed any inhibition of α-GalNAcase.

TMG-chitotriomycin as a probe for the prediction of substrate specificity of ß-N-acetylhexosaminidases.

TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was examined as a probe for the prediction of substrate specificity of ß-N-acetylhexosaminidases (HexNAcases). According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were divided into 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated for their substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides but not N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligosaccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as a molecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processing HexNAcases.

The use of rice straw broth as an appropriate medium to isolate purple nonsulfur bacteria from paddy fields

The aims were to explore an appropriate isolating medium for obtaining purple nonsulfur bacteria (PNSB) for use as biofertilizers in saline paddy fields and to obtain pure cultures. We therefore chose a defined isolating medium containing 0.25 percent NaCl, (Glutamate-Acetate broth, GA) and a rice straw broth to compare them for numbers of PNSB obtained, time to obtain pure cultures, diversity and costs. A total of 30 water and 30 sediment samples were collected from saline paddy fields in southern Thailand and used to isolate PNSB in both the isolating media. Based on 60 samples and a period of 13 days incubation under anaerobic light conditions, a greater number of samples produced PNSB growth in GA broth after only day 3; however, after that the rice straw broth provided about a 2 fold increase in the number of samples that produced PNSB growth. Colonies isolated from GA broth required a significantly higher number of repeated streaking to obtain a pure culture (average 3.5) than those from rice straw broth (average 2.7) and the latter medium also produced significantly (P < 0.05) more isolates per sample. Sixty samples of water and sediment, from rice paddies with salinity (average, 3.43 +/- 0.67 mS/cm) and slight acidity (average, pH 5.84 +/- 0.42) provided 62 PNSB isolates by GA broth and 210 isolates by rice straw broth, and rice straw broth also produced a greater prevalence of PNSB. Estimates of the costs based on current prices of media, Gas Pak and electricity to obtain PNSB with the use of GA broth was roughly 6 times higher than for the rice straw broth.

Melanogenesis inhibitors from Rabdosia japonica.

The effects of the four major ent-kaurene diterpenoids isolated from the aerial part of Rabdosia japonica (Labiatae) on murine B16-F10 melanoma cells were investigated. Among the compounds tested, oridonin and nodosin most significantly suppressed cellular melanin production when the cells were cultured with these diterpenoids. However, oridonin and nodosin exhibited cytotoxicity against the same melanoma cells with an IC(50) of 1.1 µM (0.40 µg/ml) and of 1.3 µM (0.47 µg/ml) and almost complete lethality was observed at 4.0 µM and at 8.0 µM, respectively, and therefore observed melanogenesis inhibition is mainly due to its melanocytotoxic effect. Morphological observation showed that oridonin or nodosin treated B16-F10 melanoma cells induced dendrite structure. Diterpenoids quickly formed adducts partly in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% of fetal bovine serum (10% FBS-DMEM) before their application to the cells. Approximately 20% of oridonin formed adducts within the first 15 min. Notably, dihydronodosin exhibited inferior cytotoxicity (>85% cell viability at 100 µM) but still significantly suppressed melanogenesis (>55%) when murine B16-F10 melanoma cells were cultured with this diterpenoid derivatives. Hence, dihydronodosin can be a potential melanogenesis inhibitor.

MS/MS fragmentation-guided search of TMG-chitooligomycins and their structure-activity relationship in specific ß-N-acetylglucosaminidase inhibition.

The reducing tetrasaccharide TMG-chitotriomycin (1) is an inhibitor of ß-N-acetylglucosaminidase (GlcNAcase), produced by the actinomycete Streptomyces anulatus NBRC13369. The inhibitor shows a unique inhibitory spectrum, that is, selectivity toward enzymes from chitin-containing organisms such as insects and fungi. Nevertheless, its structure-selectivity relationship remains to be clarified. In this study, we conducted a structure-guided search of analogues of 1 in order to obtain diverse N,N,N-trimethylglucosaminium (TMG)-containing chitooligosaccharides. In this approach, the specific fragmentation profile of 1 on ESI-MS/MS analysis was used for the selective detection of desired compounds. As a result, two new analogues, named TMG-chitomonomycin (3) and TMG-chitobiomycin (2), were obtained from a culture filtrate of 1-producing Streptomyces. Their enzyme-inhibiting activity revealed that the potency and selectivity depended on the degree of polymerization of the reducing end GlcNAc units. Furthermore, a computational modeling study inspired the inhibitory mechanism of TMG-related compounds as a mimic of the substrate in the Michaelis complex of the GH20 enzyme. This study is an example of the successful application of a MS/MS experiment for structure-guided isolation of natural products.

Peptide bond formation by aminolysin-A catalysis: a simple approach to enzymatic synthesis of diverse short oligopeptides and biologically active puromycins.

A new S9 family aminopeptidase derived from the actinobacterial thermophile Acidothermus cellulolyticus was cloned and engineered into a transaminopeptidase by site-directed mutagenesis of catalytic Ser(491) into Cys. The engineered biocatalyst, designated aminolysin-A, can catalyze the formation of peptide bonds to give linear homo-oligopeptides, hetero-dipeptides, and cyclic dipeptides using cost-effective substrates in a one-pot reaction. Aminolysin-A can recognize several C-terminal-modified amino acids, including the l- and d-forms, as acyl donors as well as free amines, including amino acids and puromycin aminonucleoside, as acyl acceptors. The absence of amino acid esters prevents the formation of peptides; therefore, the reaction mechanism involves aminolysis and not a reverse reaction of hydrolysis. The aminolysin system will be a beneficial tool for the preparation of structurally diverse peptide mimetics by a simple approach.

Multifunctional cytotoxic agents from Anacardium occidentale.

The effects of anacardic acids and cardols isolated from the cashew nut and apple Anacardium occidentale (Anacardiaceae) on murine B16-F10 melanoma cells were tested. Although anacardic acids and cardols were found to inhibit tyrosinase, a key enzyme in melanin synthesis, melanogenesis in melanocytes was not suppressed in cultured cells but rather enhanced. Both anacardic acids and cardols exhibited moderate cytotoxicity.

Characterization of the antiyeast compound and probiotic properties of a starter lactobacillus plantarum DW3 for possible use in fermented plant beverages

Lactobacillus plantarum DW3 produced antifungal compounds that inhibited the growth of Rhodotorula mucilaginosa DKA, contaminating yeast in fermented plant beverages (FPBs) and various potential human pathogens. Phenyllactic acid (PLA) identified by gas chromatography- mass spectrometry (GC-MS) was produced at 31 mg/L PLA in MRS medium and 5 mg/ml inhibited growth of the target yeast in vitro by 90 percent. Other inhibitors were also present but not specifically identified. Results of in vitro tests showed that DW3 also had probiotic properties as it survived various human biological barriers resistance to pH 3, bile salts, growth without vitamin B12 and the presence and absence of oxygen. Its inhibitory effect against food borne pathogenic bacteria and spoilage organisms was higher than that found for a commercial strain Lactobacillus casei R. An acute oral toxicity test on ICR mice at a high single dose of either 10(9) and 10(12) cells per mouse for 14 days showed that DW3 had no adverse effect on the general health status and there was no evidence of bacteremia. Mice fed DW3 had a reduced weight gain compared to the control. No significant difference (p > 0.05) was found for the spleen weight index (SWI) among the treatment and control groups whereas there was a significant difference (p < 0.05) for the liver weight ratio (LWR) in a group fed with 10(12) cells per mouse when compared with the control group.

Inhibition of shrimp pathogenic vibrios by extracellular compounds from a proteolytic bacterium Pseudomonas sp. W3 / Inhibición de vibrios patógenos del camarón por compuestos extracelulares de una bacterias proteolítica Pseudomonas sp.W3

Pseudomonas sp. W3, a bacterium known to produce an extracellular alkaline protease, secreted secondary metabolites that inhibited pathogenic bacteria responsible for shrimp luminous vibriosis disease. Antivibrio compounds in the culture supernatant or culture filtrates (0.45 um and 0.22 um) of the isolate W3 were tested using an agar well diffusion method on a number of pathogenic vibrios. Vibrio harveyi PSU 2015 a pathogenic isolate was the most sensitive strain. The effectiveness of preparations from the isolate W3 against V. harveyi PSU 2015, and V. cholerae PSSCMI 0062 was in the order of culture supernatant > 0.45 um culture filtrate > 0.22 um culture filtrate. These extracellular antivibrio compounds also lysed both dead and living cells of V. harveyi PSU 2015. Results of the partial characterization tests indicated that there was some particulate antivibrio compound that was destroyed by treatment with enzymes particularly alpha-chymotrypsin, autoclaving at 121ºC for 15 min and was mostly removed by filtration through a 0.22 µm filter. Most of the inhibitory compounds were of small molecular weight able to pass through a 0.22 um filter and were resistant to treatment with various enzymes, pH values between 4-8 and temperatures up to 121ºC for 30 min. The optimum pH for the antivibrio activity in the 0.45 um culture filtrate was between pH 6-7.

Pochonicine, a polyhydroxylated pyrrolizidine alkaloid from fungus Pochonia suchlasporia var. suchlasporia TAMA 87 as a potent beta-N-acetylglucosaminidase inhibitor.

A new polyhydroxylated pyrrolizidine alkaloid designated as pochonicine (1) was isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87. The structure of 1 was determined using NMR and MS techniques as (1R*, 3S*, 5S*, 6S*, 7R*, 7a S*)-5-acetamidomethyl-3-hydroxymethyl-1,6,7-trihydroxypyrrolizidine. Pochonicine (1) showed potent inhibition against beta-N-acetylglucosaminidases (GlcNAcases) of various organisms including insects, fungi, mammals, and a plant but no inhibition against beta-glucosidase of almond, alpha-glucosidase of yeast, or chitinase of Bacillus sp. The GlcNAcase inhibitory activity of pochonicine (1) was comparable to nagstatin, a potent GlcNAcase inhibitor of natural origin.

Effects of phenolic compounds isolated from Rabdosia japonica on B16-F10 melanoma cells.

Pedalitin isolated from the aerial part of Rabdosia japonica (Labiatae), exhibited cytotoxicity against the murine B16-F10 melanoma cell line with an IC(50) of 30 microm (9.5 microg/mL). As the cells were cultured with this flavone, melanin production was not suppressed, but rather enhanced. Quercetin isolated from the same source exhibited similar activities, but rutin showed neither activity.

Effects of cuminaldehyde on melanoma cells.

Cuminaldehyde (4-isopropylbenzaldehyde) suppressed melanin formation in cultured murine B16-F10 melanoma cells in a dose-dependent decrease up to 0.25 mm without affecting cell growth. Approximately 30% suppression in melanin production resulted when the cells were cultured with 0.25 mm of cuminaldehyde. This activity was not noticeable with cultured human A375 melanoma cells.