Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41.

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Microbial Production of Ursodeoxycholic Acid from Lithocholic Acid by Fusarium equiseti M41

A fungus identified as Fusarium equiseti was isolated from soil and found to carry out 7β-hydroxylation of lithocholic acid to ursodeoxycholic acid (35% yield; 350 mg/liter) in 112 h.     

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.     

Adsorption of Lithocholic Acid to Fusarium equiseti M41 as an Essential Process in Its Conversion to Ursodeoxycholic Acid

Fusarium equiseti M41 converts lithocholic acid to ursodeoxycholic acid. Adsorption of lithocholic acid particles to mycelia of F. equiseti M41 is essential in the conversion of lithocholic acid to ursodeoxycholic acid. Production of ursodeoxycholic acid was negligible when particles of lithocholic acid were absent. As the concentration of lithocholic acid particles increased, both the amount of mycelium-bound lithocholic acid and the production of ursodeoxycholic acid increased hyperbolically (K_1/2 = 1.9 g/liter and K_mapparent = 1.9 g/liter. A fluorescent lithocholic acid derivative was used to confirm that insoluble particles of lithocholic acid attached to the surface of the mycelia. The hydrophobic nature of this binding was estimated from the close relationship observed between the hydrophobicity of bile acids and their binding capacity to the mycelia. By repeated washing with 30% dimethyl sulfoxide, two binding modes of lithocholic acid were distinguished, i.e., surface binding (59% of bound lithocholic acid) and tight binding (41% of bound lithocholic acid). From the amount of tightly bound lithocholic acid, the intracellular concentration of lithocholic acid was calculated to be 1,433-fold higher than its saturating concentration in the reaction mixture, thus promoting effective conversion to ursodeoxycholic acid in the mycelia. Several lines of evidence indicated that glycoproteins of the cell wall participated in the binding of lithocholic acid.     

In vitro propagation and bioactive compound accumulation in regenerated shoots of <Emphasis Type="Italic">Dioscorea birmanica</Emphasis> Prain &amp; Burkill

Dioscorea birmanica Prain & Burkill is a Thai medicinal plant, which is often used with other medicinal plants for the treatment of cancers, AIDS, and septicemia diseases. Large numbers of this desirable plant can be produced using the plant tissue culture techniques. The objectives of this study were to investigate techniques of in vitro propagation and to examine the bioactive compounds: diosgenin-3-O-α-l-rhamnopyranosyl (1 → 2)–β-d-glucopyranoside (DBS1) content, total phenolic content, and antioxidant activity of the regenerated shoots compared to those of rhizomes growing in the field. For shoot induction, the highest numbers of shoots (2.8 ± 0.5) and nodes per shoot (5.7 ± 0.8) occurred after the single-nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg/l BA (6-benzyladenine) for 4 weeks. Shoot multiplication was achieved on MS medium supplemented with 0.01 % activated charcoal (AC) and 2 mg/l BA in combination with 0.1 mg/l IAA or 0.2 mg/l NAA. The regenerated shoots were rooted on ½ MS medium supplemented with 0.01 % AC, 2 mg/l BA and 4 mg/l NAA for 8 weeks. The survival percentage was 71.88 and small rhizomes developed after transplanting for 4–6 weeks. The quantities of 0.37 ± 0.03 % (w/w) DBS1, 44.24 ± 8.47 mg GAE/g dry extract total phenolic and DPPH radical scavenging assay with EC₅₀ value of 53.67 ± 4.16 µg/ml were determined from the regenerated shoots, while 3.27 ± 0.04 % (w/w) DBS1, 259.67 ± 7.34 mg GAE/g dry extract total phenolic and DPPH radical scavenging assay with EC₅₀ value of 11.42 ± 3.28 µg/ml were found in the mother rhizomes.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.     

Animal Models for Echinostoma malayanum Infection: Worm Recovery and Some Pathology

Echinostomes are intestinal trematodes that infect a wide range of vertebrate hosts, including humans, in their adult stage and also parasitize numerous invertebrate and cold-blooded vertebrate hosts in their larval stages. The purpose of this study was to compare Echinostoma malayanum parasite growth, including worm recovery, body size of adult worms, eggs per worm, eggs per gram of feces, and pathological changes in the small intestine of experimental animals. In this study, 6-8-week-old male hamsters, rats, mice, and gerbils were infected with echinostome metacercariae and then sacrificed at day 60 post-infection. The small intestine and feces of each infected animal were collected and then processed for analysis. The results showed that worm recovery, eggs per worm, and eggs per gram of feces from all infected hamsters were higher compared with infected rats and mice. However, in infected gerbils, no parasites were observed in the small intestine, and there were no parasite eggs in the feces. The volume of eggs per gram of feces and eggs per worm were related to parasite size. The results of histopathological changes in the small intestine of infected groups showed abnormal villi and goblet cells, as evidenced by short villi and an increase in the number and size of goblet cells compared with the normal control group.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

Direct fermentation of l(+)-lactic acid from cassava pulp by solid state culture of Rhizopus oryzae

This study shows that Rhizopus oryzae is capable of directly utilizing cassava pulp alone to L: -lactic acid in solid state fermentation (SSF). pH control at 6.0 helped prevent end product inhibition. Increasing lactate titer was observed at the higher initial moistened water due to the higher degree of substrate swelling and hydrolysis. With shaking, limited ethanol production but no change in lactate titer was observed. Rigorous shaking gave better oxygen transfer but presumably caused cell damage leading to substrate utilization through the biosynthesis route. Supplementing cassava pulp with nitrogen enhanced growth but not lactate production. Under the optimal conditions, R. oryzae converted the sole cassava pulp into lactic acid at the titer of 206.20?mg per g initial dry pulp. With the help of commercial cellulase and glucoamylase, the dramatically increasing lactate titer of 463.18?mg per g initial dry pulp was achieved via SSF.