Endo-ß-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

The crystal structure of wild-type endo-ß-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (ß/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions.

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines. The aim of this work was the transcriptional characterization of six O. oeni strains, four of them selected from distinct winemaking regions of Portugal, as candidates to malolactic starters, and two commercial malolactic starters. Using crossed assays with wines from different Portuguese winemaking regions, strain characteristic transcriptional patterns induced by each wine were analyzed based on Random Arbitrarily Primed PCR (RAP-PCR). The obtained results suggest that the starter strains showed more constrained and limited transcription profiles, whereas a high variation on the distribution of the transcription profiles was observed for the regional strains in each wine. According with our results, RAP-PCR is a useful technique for a preliminary investigation of strain behavior under different wine environmental conditions, which can be applied in field studies to monitor differential patterns of global gene expression and to select markers for the surveillance of malolactic starters performance in winemaking, as well as for quality and safety control.

A novel molecular method for identification of Oenococcus oeni and its specific detection in wine.

Oenococcus oeni is a species of lactic acid bacteria with economic interest in winemaking. Using both in silico and in vitro analyses, a molecular method was developed that allows the identification of O. oeni isolates and its detection from wine samples. The method is based on the amplification of 16S rRNA gene with universal primers followed by restriction with the endonuclease FseI, generating two fragments of 326 and 1233 bp. Among wine bacteria, the FseI recognition sequence is only found in the 16S rRNA gene of O. oeni, ensuring the specificity of the method. The use of Whatman FTA cards for DNA extraction and purification is an efficient and interesting alternative to current methods, as samples can be easily collected at wineries by a non-specialized technician, stored at room temperature and sent in a mail envelope to the analytical laboratory for processing. The proposed method, with a detection limit between 10(2) and 10(3) cfu/mL and a full turnaround time of ca. 8h, ensures the rapid and reliable detection of O. oeni in wine samples during winemaking surveillance and wine quality control.

Penicillium glabrum cork-colonising isolates - preliminary analysis of their genomic similarity.

The cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are not completely understood although they are considered to be involved in the so-called "cork taint" of wine. It is essential to (a) identify environmental constraints which define the appearance of the colonising fungal species and (b) trace their origin to the forest and/or the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was confirmed, followed by a high diversity of Penicillium species. Penicillium glabrum, which was found in all samples, was the most frequently isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork specific fungi can contribute.