Characterization of an ethanol-tolerant 1,4-ß-xylosidase produced by Pichia membranifaciens.

AIMS: The purification and biochemical properties of the 1,4-ß-xylosidase of an oenological yeast were investigated. METHODS AND RESULTS: An ethanol-tolerant 1,4-ß-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50kDa by SDS-PAGE. The activity of 1,4-ß-xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48±0·06mmol l(-1) and a Vmax of 7·4±0·1µmol min(-1)mg(-1) protein for p-nitrophenyl-ß-d-xylopyranoside. CONCLUSIONS: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may be useful for assessing the ability of the 1,4-ß-xylosidase from P. membranifaciens to be used in the bioethanol production process.

Characterization of glycolytic activities from non-Saccharomyces yeasts isolated from Bobal musts.

A large number of non-Saccharomyces yeasts were isolated from grapes of Bobal variety and identified according to their physiological and molecular characteristics. The yeasts were tested to determine the presence of ß-glucosidase, ß-xylosidase, α-arabinosidase, and α-rhamnosidase activities and five isolates were selected. All enzymatic activities were induced by the presence of glycosides as the only carbon source in the medium, which seems to be a characteristic of the yeast isolate, and were characterized according to different parameters of enological interest.

The use of alternative technologies to develop malolactic fermentation in wine.

The development of the malolactic fermentation, bioconversion of L-malic acid to L-lactic acid, is a difficult and time-consuming process that does not always proceed favorably under the natural conditions of wine. Traditional fermentations are used worldwide to produce high-quality wines, although delay or failure is not an unusual outcome. During recent years several technologies have been proposed to induce biological deacidification of wines by using malolactic bacteria, principally Oenococcus oeni and Lactobacillus sp. These alternative technologies usually involve the use of high densities of cells or enzymes, free or immobilized onto different matrices. Immobilization materials, several types of bioreactors, and the properties of many specific systems are discussed in this review.

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni.

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.

The ribosomal DNA of the Zygomycete Mucor miehei.

The ribosomal DNA-from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70-80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi.

Expression of YWP1, a gene that encodes a specific Yarrowia lipolytica mycelial cell wall protein, in Saccharomyces cerevisiae.

The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids. Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 5' noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S. cerevisiae promoters. A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants. Ywp1 processing and incorporation to the cell wall were similar in both Y. lipolytica and S. cerevisiae but not in its final localization in the cell wall. In Y. lipolytica Ywp1 is covalently bound to the cell wall (it is released only by Zymolyase digestion), whereas in S. cerevisiae it was not (it was released by boiling SDS solutions). These results suggest that the sequences involved in recognition, anchoring of a protein to the cell wall, or the catalytic activities implicated are different, at least for Ywp1, in Y. lipolytica and S. cerevisiae. Another possibility is that the target for attachment of Ywp1 is missing or cryptic in the cell wall of S. cerevisiae.