Biomedical subjects

F Randez-Gil

Publications and source records attributed to F Randez-Gil.

14 recordsLinked to original sources

PubMed2003

Osmotolerance and leavening ability in sweet and frozen sweet dough. Comparative analysis between Torulaspora delbrueckii and Saccharomyces cerevisiae baker's yeast strains.

The response of Saccharomyces cerevisiae and freeze-tolerant Torulaspora delbrueckii strains to osmotic stress and their CO2 production capacity in sweet and frozen-sweet dough has been examined. T. delbrueckii strains, IGC5321 and IGC5323 showed higher leavening ability than Saccharomyces, specially after exposure to hyperosmotic stress of bread dough containing 20% sucrose and 2% salt added. In addition, Torulaspora and especially T. delbrueckii IGC5321 exhibited no loss of CO2 production capacity during freeze-thaw stress. Overall, these results appeared to indicate that Torulaspora cells are more tolerant than Saccharomyces to osmotic stress of bread dough. This trait correlated with a low invertase activity, a slow rate of trehalose mobilisation and the ability to respond rapidly to osmotic stress. Growth behaviour on high osmotic synthetic media was also examined. Cells of the IGC5321 strain showed intrinsic osmotolerance and ion toxicity resistance. However, T. delbrueckii IGC5323 exhibited a clear phenotype of osmosensitivity. Hence, this characteristic may not be essential or the only determinant for leavening ability in salted high-sugar dough.

Bread↗

PubMed2000

Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans.

Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.

Aspergillus nidulans↗

PubMed1999

Engineering baker's yeast: room for improvement.

Bread making is one of the oldest food-manufacturing processes. However, it is only in the past few years that recombinant-DNA technology has led to dramatic changes in formulation, ingredients or processing conditions. New strains of baker's yeast that produce CO2 more rapidly, are more resistant to stress or produce proteins or metabolites that can modify bread flavour, dough rheology or shelf-life are now emerging.

Cellulose↗

PubMed1998

Hexokinase PII has a double cytosolic-nuclear localisation in Saccharomyces cerevisiae.

We describe here that the HXK2 gene product, isoenzyme PII of hexokinase, is located in both the nucleus and the cytoplasm of Saccharomyces cerevisiae cells. This conclusion is supported by assays of hexokinase-specific activity in isolated nuclei from wild-type and hxk1lhxk2 double mutant strains, by immunoblot experiments using anti-Hxk2 antibodies and by observation of the fluorescence distribution of a Hxk2-GFP fusion protein in cells transformed with the HXK2::gfp gene.

Cell Fractionation↗

PubMed1998

Yeast Clk-1 homologue (Coq7/Cat5) is a mitochondrial protein in coenzyme Q synthesis.

Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans. The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation. We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration. These results obtained in the yeast model suggest that the effects on development and life span in C. elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant.

Amino Acid Sequence↗

PubMed1998

Carbon source-dependent phosphorylation of hexokinase PII and its role in the glucose-signaling response in yeast.

The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PH is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, and cid1/glc7. Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells with lambda-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2 (S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.

Adaptation, Biological↗

PubMed1997

Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p.

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

Catalase↗

PubMed1995

DOGR1 and DOGR2: two genes from Saccharomyces cerevisiae that confer 2-deoxyglucose resistance when overexpressed.

Saccharomyces cerevisiae contains two genes (DOGR1 and DOGR2) that are able to confer 2-deoxyglucose resistance when they are overexpressed. These genes are very similar, sharing 92% identity at the protein level. They code for two isoenzymes with 2-deoxyglucose-6 phosphate (2-DOG-6P) phosphatase activity. These enzymes have been purified and characterized. DogR1p shows an optimum pH of 6, an optimum temperature of 30 degrees C and a KM on 2-DOG-6P of 17 mM. DogR2p shows a similar optimum pH, but the optimum temperature is 40 degrees C and it exhibits a KM on 2-DOG-6P of 41 mM. Both enzymes require 10 mM-MgCl2 for maximal activity and they are inhibited by inorganic phosphate.

Alleles↗

PubMed1995

The expression of a specific 2-deoxyglucose-6P phosphatase prevents catabolite repression mediated by 2-deoxyglucose in yeast.

2-deoxyglucose (2-DOG), a non-metabolize analogue of glucose, is taken up by yeast using the same transporter(s) as glucose and is phosphorylated by hexokinases producing 2-deoxyglucose-6-P. We found that in DOGR yeasts, 2-DOG was not able to trigger glucose repression, even at concentrations of 0.5%. This result suggests that the specific 2-DOG-6P phosphatase, the enzyme responsible for the DOGR phenotype, may be involved in inhibiting the process of catabolite repression mediated by 2-DOG.

Deoxyglucose↗

PubMed1994

Nucleotide sequence of a putative peroxisomal protein from the yeast Lipomyces kononenkoae.

A cDNA corresponding to a putative peroxisomal protein from the yeast Lipomyces kononenkoae has been isolated. It contains an ORF of 498 bp that predicts a polypeptide of 166 amino acids with an apparent molecular mass of 17,365 Da. The protein shares a high degree of homology with proteins associated to the peroxisomal membrane of Candida boidinii.

Amino Acid Sequence↗

PubMed1994

Molecular characterization of a gene that confers 2-deoxyglucose resistance in yeast.

We have isolated a gene whose expression enables yeast cells to overcome the inhibition of growth produced by the presence of 2-deoxyglucose. The gene contains an open reading frame of 738 bp that may code for a protein of 27,100 Da. Cells carrying this gene contain high levels of a specific 2-deoxyglucose-6-phosphate phosphatase. The expression of this phosphatase is increased by the presence of 2-deoxyglucose and is constant along the growth curve.

Amino Acid Sequence↗

PubMed1993

Expression of Aspergillus oryzae alpha-amylase gene in Saccharomyces cerevisiae.

A fragment containing the full length cDNA from Aspergillus oryzae alpha-amylase has been amplified by PCR using specific synthetic oligonucleotides. The amplified cDNA was designed to favour its expression in yeast by modifying its upstream untranslated region. It was subcloned in the expression vector pYEX alpha 1, placed under the control of the yeast CYC1-GAL10 promoter and used to transform Saccharomyces cerevisiae. Cells were then able to express and secrete active alpha-amylase to the medium in a regulated fashion. The recombinant enzyme had similar electrophoretic mobility and catalytic properties to the original A. oryzae alpha-amylase.

Aspergillus oryzae↗

PubMed1991

Direct derivative spectrophotometric determination of nitrazepam and clonazepam in biological fluids.

The use of fifth order derivative spectra allows the direct determination of nitrazepam in urine at 388 nm with a limit of detection of 1 microgram ml-1. The determination of nitrazepam in blood plasma can be carried out directly by measurement at 402 nm in the fourth order derivative spectra with a limit of detection of 1.5 micrograms ml-1. Clonazepam can be determined directly in urine samples at 384 nm by using sixth order derivative spectra with a limit of detection of 1 microgram ml-1 and in blood plasma by using fourth order derivative spectra at 384 nm with a limit of detection of 0.5 ppm.

Clonazepam↗

PubMedDate not supplied

Purification and characterization of a new alpha-amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae.

A new alpha-amylase from the extracellular culture of the yeast Lipomyces kononenkoae CBS 5608 has been purified to homogeneity by ammonium sulphate treatment, affinity binding on cross-linked starch, and DEAE-Biogel A chromatography. The enzyme was monomeric, with an apparent M(r) of 76 kilodaltons, pI < 3.5, and optimum pH 4.5-5.0, and exhibited intermediate thermal stability. The temperature for optimal enzyme activity was 70 degrees C. It is a glycoprotein with both N- and O-linked sugars. Kinetic analyses indicate that the enzyme has an endoamylolytic mechanism. The kM for soluble starch was 0.80 g.L-1 and the kcat was 622.s-1.

Enzyme Stability↗