Biomedical subjects

M Asther

Publications and source records attributed to M Asther.

34 records · Page 2Linked to original sources

PubMed1999

Basidiomycetes as new biotechnological tools to generate natural aromatic flavours for the food industry

Consumer preference for natural food additives has led to an increasing demand for natural aromatic compounds. An alternative production process to plant and chemical sources is the use of biotechnological methods involving microorganisms, which ensure a stable supply, quality and price. Among filamentous fungi, white-rot basidiomycetes represent an important group that generate a wide range of flavouring compounds, particularly aromatic molecules. Their biotechnological potential to produce natural aromatic flavours de novo or by biotransformation thus presents a very interesting challenge.

Journal Article↗

PubMed1999

Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium.

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.

Microscopy, Atomic Force↗

PubMed1999

Enhanced benzaldehyde formation by a monokaryotic strain of Pycnoporus cinnabarinus using a selective solid adsorbent in the culture medium.

A monokaryotic strain of the white-rot fungus Pycnoporus cinnabarinus was shown to produce, in a 2-L bioreactor culture, 100 mg.L-1 benzaldehyde (bitter almond aroma) from L-phenylalanine with a productivity of 33 mg.L-1.day-1. The addition of HP20 resin, a styrene divinylbenzene copolymer highly selective for benzaldehyde, enabled an eightfold increase in the production of benzaldehyde and a twofold increase in productivity. In the presence of HP20 resin, the production of 790 mg.L-1 benzaldehyde was concomitant with the synthesis of cinnamic acid derivatives of high organoleptic notes such as cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate.

Adsorption↗

PubMed1998

Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae.

The subcellular localization of the phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) of Aspergillus oryzae was investigated using Western blot analysis of the cell protein extracts, a cellular membrane fractionation technique, and transmission electron microscopy. The PG/PI-TP, as detected by Western blot analysis with a specific immune serum, was found to be mainly cytoplasmic and partly associated with intracellular membranes. A fractionation experiment was conducted after homogenization of the filamentous fungus mycelium. The endoplasmic reticulum, Golgi-like vesicles, and the plasma membrane were separated by isopycnic ultracentrifugation on a sucrose gradient, and our data revealed that the immunodetected PG/PI-TP was only associated with the Golgi-like apparatus. All these results were documented by electron microscopy and indicate here for the first time that there exists a specific phospholipid transfer protein in a filamentous fungus that is localized in the cytoplasm and associated with Golgi-like vesicles.

Animals↗

PubMed1996

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus.

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg1-1 with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg1-1 with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.

Aspergillus niger↗

PubMed1995

Purification and characterization of a novel specific phosphatidylglycerol-phosphatidylinositol transfer protein with high activity from Aspergillus oryzae.

A novel phospholipid transfer protein has been purified to homogeneity 406-fold from the filamentous fungus Aspergillus oryzae. The successive steps of purification comprised ultrafiltration, gel filtration on Sephadex G-75, ion exchange chromatographies on DEAE-Sepharose and Mono Q. The active protein is a monomer with a molecular mass of 19,000, estimated from SDS electrophoresis, amino acid composition as well as gel filtration. The isoelectric point is 4.8. The amino acid composition is characterized by a high amount of Gly, Leu, Ser, Asx and Glx residues and 4 Cys residues. N-terminal sequence was determined and compared with M. mucedo sequence. The purified protein was found to transfer preferentially phosphatidylglycerol and phosphatidylinositol over phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine and no phosphatidic acid. Optimal temperature for in vitro transfer was 25-30 degrees C and optimal pH 4-7. Heating protein at 100 degrees C does not inactivate protein whereas a denaturation with urea is irreversible.

Amino Acid Sequence↗

PubMed1994

Biotransformation of the Herbicide Atrazine by the White Rot Fungus Phanerochaete chrysosporium.

Biotransformation of atrazine by the white rot fungus Phanerochaete chrysosporium was demonstrated by a 48% decrease of the initial herbicide concentration in the growth medium within the first 4 days of incubation, which corresponded to the mycelium-growing phase. Results clearly established the mineralization of the ethyl group of the herbicide. Analysis of the growth medium showed the formation of hydroxylated and/or N-dealkylated metabolites of atrazine during fungal degradation.

Journal Article↗

PubMed1994

Filamentous fungi with high cytosolic phospholipid transfer activity in the presence of exogenous phospholipid.

The phospholipid transfer activity of cell extracts from 15 filamentous fungus strains grown on a medium containing phospholipids as the carbon source was measured by a fluorescence assay. This assay was based on the transfer of pyrene-labeled phosphatidylcholines forming the donor vesicles to acceptor vesicles composed of egg phosphatidylcholines. The highest phosphatidylcholine transfer activity was obtained with cell extracts from Aspergillus oryzae. The presence of exogenous phospholipids in the culture medium of A. oryzae was shown to increase markedly the activity of phospholipid transfer as well as the pool of exocellular proteins during the primary phase of growth. Modifications in the biochemical marker activities of cellular organelles were observed: succinate dehydrogenase, a mitochondrial marker; inosine diphosphatase, a Golgi system marker; and cytochrome c oxidoreductase, an endoplasmic reticulum marker, were increased 7.3-, 2-, and 22-fold, respectively, when A. oryzae was grown in the presence of phospholipids.

Journal Article↗

PubMed1994

Detection of rodlets in the outer wall region of conidiospores of Phanerochaete chrysosporium.

The surface morphology of the conidiospores of Phanerochaete chrysosporium was investigated using freeze-etching. A multilayered structure composed of rodlets was detected. The rodlets had a diameter of 10.2 +/- 0.5 nm and were organised as long parallel fibres. Granules, smooth materials, and bark-like structures were found to cover part of this rodlet layer. During germination, the outer pellicle of the spore wall became fragmented and residual aggregates with rodlets were disseminated on the new conidiospore surface. The latter, as well as the germ tube, was composed of fibrillar material.

Basidiomycota↗

PubMed1993

In situ localization of the secretion of lignin peroxidases in colonies of Phanerochaete chrysosporium using a sandwiched mode of culture.

Protein secretion and growth were investigated in Phanerochaete chrysosporium by using cultures sandwiched between perforated polycarbonate membranes. Labelling of colonies with radioactive N-acetylglucosamine and L-methionine indicated a close correlation between growth and general protein secretion, even in a central area of the colony secreting the idiophase enzymes lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP). Comparison of the sites of release into the medium of newly synthesized proteins and immuno-detected lignin peroxidases suggested that diffusion of the enzymes from the walls was a limiting step in the release of peroxidases into the medium. Microautoradiography of colonies exposed to N-acetyl[3H]glucosamine revealed the apical growth of thin hyphae and branches (4 to 5 microns diameter on average) in the central secreting area. These secondary hyphae showed peroxidase activity and reacted with lignin peroxidase antibodies. Although it was not possible to directly visualize secretion at hyphal tips, the results suggest that peroxidases (LiP and MnP) are initially secreted at the apex of secondary growing hyphae and later slowly released into the surrounding medium.

Fungi↗

PubMed1993

Spatial and temporal accumulation of mRNAs encoding two common lignin peroxidases in Phanerochaete chrysosporium.

Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1) and lignin peroxidase (LiP H8) mRNAs indicated that the expression of MnP-1 and Lip H8 genes started simultaneously in the central area of 3-day-old colonies. With time the signals for both transcripts spread to more-peripheral areas while decreasing in intensity. Furthermore, the appearance of MnP protein, as detected with specific immune serum, immediately followed accumulation of the MnP-1 mRNA transcript. However, LiP protein could be detected only some time after accumulation of LiP H8 mRNA.

Cell Compartmentation↗

PubMed1993

Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation.

The conidiospores of the white rot basidiomycete Phanerochaete chrysosporium tend to aggregate during swelling and germination in agitated liquid medium; as time passes, the initial aggregates tend to associate together and to capture conidiospores that remain isolated. The surface chemical compositions of the conidiospores and of developed hyphae were analyzed by X-ray photoelectron spectroscopy. The data were interpreted by modelling the surface in terms of proteins, polysaccharides and hydrocarbonlike compounds. The surface molecular composition of the dormant conidiospores was estimated to be about 45% proteins, 20% carbohydrates, and 35% hydrocarbonlike compounds. There was an increase in the polysaccharide content during germination. Later, when the hyphae were developed, the polysaccharide content became still higher, and the protein content dropped. The initial step of aggregation is attributed to polysaccharide bridging; its occurrence cannot be explained by a change of the overall hydrophobicity or electrical properties of the conidiospores.

Carbon↗

PubMed1992

An improved method for the purification of lignin peroxidases from Phanerochaete chrysosporium INA-12: properties of two major isoforms.

1. Phanerochaete chrysosporium INA-12 secretes several lignin peroxidase isoenzymes. This paper reports an improved procedure for the purification of the different isoforms compared to those previously described. 2. Lignin peroxidases are first concentrated and prefractionated on fast-flow ion-exchangers which avoid concentration by ultrafiltration and dialysis. 3. Further purification is achieved by hydrophobic interaction chromatography and anion-exchange FPLC. 4. Two major forms were purified to homogeneity. Kinetic measurements and protein characterization (isoelectric points, phosphate content) suggest that they are similar to those produced by P. chrysosporium BKM strain.

Basidiomycota↗

PubMed1990

Characterization of Peroxidase Secretion and Subcellular Organization of Phanerochaete chrysosporium INA-12 in the Presence of Various Soybean Phospholipid Fractions.

Stimulation of lignin peroxidase production by exogenous phospholipids depends on the composition of the phospholipid fraction prepared by using the Nattermann process. The fraction composed mainly of negatively charged phospholipids (NAT 89) was the most efficient source for exoprotein secretion by Phanerochaete chrysosporium INA-12. The results of biochemical marker assays and ultrastructural morphology determination by electron microscopy were correlated. Activities of succinate dehydrogenase, a mitochondrial marker, and cytochrome c oxidoreductase, an endoplasmic reticulum (ER) marker, were increased 1.3- and 2.2-fold, respectively, in the presence of NAT 89. Electron microscopy observations suggested that the amount of mitochondria and ER in culture containing phospholipids was increased at the optimum day of lignin peroxidase production. Therefore, phospholipids enhanced energetic metabolism of strain INA-12 and markedly modified fungus physiology. Since ER is involved in enzyme synthesis, we suggest that its increased amount in mycelium cultured with NAT 89 is directly associated with the higher production of lignin peroxidase.

Journal Article↗

PubMed1988

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting.

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37 degrees C, is for the mycelium-growing phase; the other, at 30 degrees C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30 degrees C for the entire fermentation period or when cultures were grown at 37 degrees C for the first 2 days of incubation and then shifted to 30 degrees C, compared with the activity of control cultures grown at 37 degrees C for the entire fermentation period. The unsaturation of fatty acid (Delta/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40 degrees C.

Journal Article↗

PubMedDate not supplied

Immobilization as a tool for the stabilization of lignin peroxidase produced by Phanerochaete chrysosporium INA-12.

Lignin peroxidase immobilization was achieved by covalent coupling on CNBr-Sepharose 4B. Protein immobilization yield was around 80%. For veratryl alcohol oxidation, in the presence of hydrogen peroxide, both soluble and bound enzymes exhibited the same pH profile with an optimum near 2.5. Catalytic parameters (kc and Km) were seriously affected by immobilization. On the other hand, immobilization provided a noticeable stabilization of the enzyme against acidic pH and high temperatures. A 15-20 increase in the half-inactivation times at pH 2.2 and 2.7, respectively, could be observed. Bound enzyme was also much more thermostable than soluble.

Basidiomycota↗