Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation.

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (ß-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15-18days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. ß-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for ß-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.

alpha-Glucosidase inhibitory activity of triterpenoids from Cichorium intybus.

Two new triterpenoids, 18alpha,19beta-20(30)-taraxasten-3beta,21alpha-diol (cichoridiol) (1) and 17-epi-methyl-6-hydroxyangolensate (intybusoloid) (2), were obtained from the methanolic extract of seeds of Cichorium intybus along with 11 known compounds, lupeol (3), friedelin (4), beta-sitosterol (5), stigmasterol (6), betulinic acid (7), betulin (8), betulinaldehyde (9), syringic acid (10), vanillic acid (11) 6,7-dihydroxycoumarin (12), and methyl-alpha-D-galactopyranoside (13). Compounds 1, 1a, and 11 showed a good alpha-glucosidase inhibitory activity.

Inducible nitric oxide synthase involvement in the mechanism of action of Saccharomyces boulardii in castor oil-induced diarrhoea in rats.

The biotherapeutic agent Saccharomyces boulardii has been shown to inhibit castor oil-induced diarrhoea in rats in a dose-response fashion, and one of the suggested mechanisms of action included involvement of the nitric oxide pathway. The present study was designed to address this mechanism of action by firstly measuring the effects of S. boulardii on the inducible nitric oxide synthase (iNOS) isoform activity in vitro. Second, the effects of S. boulardii on the increase in colonic citrulline level associated with castor oil treatment were examined. In vitro, S. boulardii showed a dose-dependent inhibition of iNOS activity with an IC50 of 0.89 mg/ml. In the rat diarrhoea model, the antidiarrhoeal effect of S. boulardii was confirmed using a single oral dose of 12 x 10(10) CFU/kg (viable cells). In this model, castor oil significantly elevated citrulline level from 2526+/-164 to 3501+/-193 nmol/g in the colon. When the rats were treated with the same antidiarrhoeal single dose of S. boulardii, no increase in citrulline level was observed. Moreover, the iNOS inhibitor 1400 W at 10 mg/kg and the inhibitor of iNOS expression dexamethasone at 1 mg/kg, administered subcutaneously, blocked the citrulline production induced by the laxative. Taken together, these findings confirm the involvement of inhibition of the inducible isoform of nitric oxide synthase in the mechanism of action of S. boulardii in diarrhoea.

Optimization of an Escherichia coli system for cell-free synthesis of selectively N-labelled proteins for rapid analysis by NMR spectroscopy.

Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces.

Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-specific polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been amplified and used to construct gene fusions with the beta-galactosidase gene. On the basis of beta-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and filament formation conditions.

Pharmacogenetics of cyclic guanylate, antioxidants, and antioxidant enzymes in Saccharomyces.

Supplements of antioxidants, superoxide dismutase (SOD), catalase, cyclic guanylate (cGMP), and theophylline, or omission of iron and copper from the medium are therapeutic for the inferior growth and viability of yeast mutants doubly deficient in mitochondrial and exocellular SOD isozymes under oxidative stresses. Cyclic adenylate tends to be ineffective or counterproductive. Oxy-stress resistant revertants are cross-resistant to other oxy-stresses and acquire one, the other, or both isozymes. The principal conclusions are: i) a genetic defect in cGMP metabolism probably compromises regulation of the enzymes' synthesis; ii) the enzymes are only essential for growth and viability under oxidative stresses; iii) oxidative toxicity is mediated by both exo- and endocellular oxy-radicals, particularly hydroxyl radicals; and iv) the pharmacogenetic features and the mutants' phenotypes are quite similar to those of negative antioxidant enzyme regulatory mutants of the related ascomycete Neurospora.

The effect of myo-inositol deficiency on phosphatases of yeast.

Activities of several phosphohydrolases are significantly enhanced when cells of the inositol-requiring yeast, Saccharomyces uvarum ATCC 9080, are deprived of inositol. This effect is most pronounced for the external acid phosphatase and cannot be explained simply by limitation of cellular growth, because starvation for vitamins or sulphate has no effect on acid phosphatase activities. Excessive secretion of acid phosphatase by spheroplasts prepared from inositol-deficient cells is greatly reduced when the spheroplast medium is supplemented with inositol and is immediately suppressed by the addition of cycloheximide. These results together with data obtained from experiments with whole cells, employing cycloheximide and actinomycin D, point to a regulatory effect of inositol limitation at the level of transcription. The external enzymes beta-D-fructofuranosidase, alpha-D-galactosidase and L-asparaginase, and the vacuolar enzyme carboxypeptidase Y are not affected by inositol deficiency indicating that inositol deficiency has no general effect on protein secretion.

The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis.

The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-biphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4]. In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities. The thermostability of phosphofructokinase (at 53 degrees C) increased, while that of aldolase (at 48 degrees C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52 degrees C) was also unchanged.

Pectinolytic activity of Saccharomyces fragilis cultured in controlled environments.

Seven of 30 yeast stock cultures, covering nine genera, and 13 of 39 yeasts isolated from grapes gave positive reactions when screened for pectinolytic activity on pectin gel plates. The seven stock cultures covered six species and four genera. Only one of the yeasts, Saccharomyces fragilis Y49, excreted discernible pectinolytic activity into the fluid of shake flask cultures; the activity was partially constitutive and was repressed by high oxygen tensions.

Biosynthesis of glutamic acid in Saccharomyces: accumulation of tricarboxylic acid cycle intermediates in a glutamate auxotroph.

Aconitaseless glutamic acid auxotroph MO-1-9B of Saccharomyces grew in glutamic acid-supplemented minimal medium, but failed to grow when glutamic acid was substituted by proline, arginine, ornithine, or glutamine. This mutant was also unable to utilize lactate or glycerol as a carbon source. Under a glutamic acid-limiting condition, by using acetate-1-(14)C as tracer, the mutant accumulated rather large amounts of (14)C-citric acid and (14)C-succinic acid when compared with the wild-type strain. Under excess glutamic acid supplementation, accumulation of citric acid and succinic acid was considerably reduced. When (14)C-glutamic acid-(U) was used as tracer, (14)C-alpha-ketoglutaric acid, (14)C-citric acid, and (14)C-succinic acid were accumulated in the mutant. The citric acid peak was the largest, followed by alpha-ketoglutaric acid and succinic acid. In the wild-type strain under similar conditions, only small amounts of (14)C-citric acid and (14)C-succinic acid and no (14)C-alpha-ketoglutaric acid were accumulated.

Fermenting yeasts associated with softening and gas-pocket formation in olives.

Fermenting, pectolytic yeasts were isolated from a massive commercial outbreak of softening and gas-pocket formation in olives that had been stored in acidified, low-salt brines in an attempt to reduce the problem of brine disposal. The suspected yeasts represented three different species: Saccharomyces oleaginosus, S. kluyveri, and Hansenula anomala var. anomala. All pectolytic cultures produced pectin esterase and polygalacturonase but no pectic acid trans-eliminase when grown in nutrient glucose broth. Crude, cell-free dialyzed enzyme preparations measured viscosimetrically exhibited optimal activity on sodium polygalacturonate at pH 6.0 and 45 C and were active in the range of pH 4.0 to 9.0 and 10 to 60 C.

New mutant types at the ade3 locus of Saccharomyces cerevisiae.

Six mutants, allelic to ade3, were isolated after mutagenic treatment of a prototrophic strain of yeast. All six grow on medium supplemented with adenine alone and four respond to histidine. Supplementation with adenine plus histidine or methionine inhibits growth, but a mixture of these three is stimulatory. Heteroallelic diploids formed by the new mutants with the standard ade3 can resemble either parent or show an intermediate phenotype. The new mutants, unlike standard ade3, are not fully epistatic to ade2. The activities of three enzymes concerned in tetrahydrofolate metabolism have been assayed in the new and standard ade3 mutants and wild type. The only difference detected between the new and standard ade3 was in the levels of 10-formyltetrahydrofolate synthetase. Activity in the new mutants ranged from 36 to 109% of wild type compared with 10 to 12% in the standard ade3. Possible mechanisms to account for the varied phenotypes at the ade3 locus are discussed.

Galactose transport in Saccharomyces cerevisiae. II. Characteristics of galactose uptake and exchange in galactokinaseless cells.

The characteristics of the inducible galactose system in Saccharomyces cerevisiae were studied by using the nonmetabolized galactose analogues, l-arabinose and d-fucose, and galactokinaseless and transportless mutants. Induced wild-type cells transport l-arabinose by facilitated diffusion. Transportless cells transport neither galactose nor l-arabinose above the noninduced rate, whereas galactokinaseless cells transport galactose l-arabinose and d-fucose by facilitated diffusion. Determination of unidirectional rate of (14)C-labeled galactose uptake by preloaded galactokinaseless cells, containing a large unlabeled free-galactose pool, showed that the rate of galactose uptake by facilitated diffusion is greater than the rate of galactose metabolism at similar external galactose concentrations.