An ␣-xylosidase active against xyloglucan oligosaccharides was purified from cabbage (Brassica oleracea var. capitata) leaves. Two peptide sequences were obtained from this protein, the N-terminal and an internal one, and these were used to identify an Arabidopsis gene coding for an ␣-xylosidase that we propose to call AtXYL1. It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM. AtXYL1 comprised three exons and encoded a peptide that was 915 amino acids long, with a potential signal peptide of 22 amino acids and eight possible N-glycosylation sites. The protein encoded by AtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only ␣-xylosidases, but also ␣-glucosidases. The ␣-xylosidase activity is present in apoplastic extractions from Arabidopsis seedlings, as suggested by the deduced signal peptide. The first eight leaves from Arabidopsis plants were harvested to analyze ␣-xylosidase activity and AtXYL1 expression levels. Both increased from older to younger leaves, where xyloglucan turnover is expected to be higher. When this gene was introduced in a suitable expression vector and used to transform Saccharomyces cerevisiae, significantly higher ␣-xylosidase activity was detected in the yeast cells. ␣-Glucosidase activity was also increased in the transformed cells, although to a lesser extent. These results show that AtXYL1 encodes for an apoplastic ␣-xylosidase active against xyloglucan oligosaccharides that probably also has activity against p-nitrophenyl-␣-d-glucoside.
Saccharomyces cerevisiae CECT1389 secreted an extracellular endopolygalacturonase (EC 3.2.1.15) when grown in shake flasks in medium containing galactose alone, or either galactose and polygalacturonic acid or galactose and galacturonic acid as the carbon sources. The synthesis of the enzyme was repressed by glucose and by high oxygen tensions. The enzyme was partially purified by gel exclusion chromatography over Sephacryl S-200, where it showed an apparent molecular mass of 39 kDa; the value determined by high-performance liquid chromatography (HPLC) was 65 kDa. The optimal temperature and pH for enzyme activity were 45 degrees C and 5.5, respectively. The Km and Vmax values for polygalacturonic acid were 4.7 mg.mL-1 and 6.4 nmol.mL-1.min-1. The Ki for HgCl2 was 6.8 x 10(-5) M. The enzyme exhibited an endo-splitting mechanism as deduced from viscosimetry experiments as well as from an HPLC study of the end products.
When grown in the appropriate medium, several yeast species produce pectinases able to degrade pectic substances. It is mainly exocellular endopolygalacturonases that break pectins or pectate down by hydrolysis of alpha-1,4-glycosidic linkages in a random way. Biochemical characterisation of these enzymes has shown that they have an optimal pH in the acidic region and an optimal temperature between 40 and 55 degrees C. Their production by yeasts is a constitutive feature and is repressed by the glucose concentration and aeration. Pectic substances and their hydrolysis products are used as carbon sources by a limited number of yeasts and hence these enzymes must be involved in the colonisation of different parts of plants, including fruits. The first yeast pectic enzyme (encoded by the PSE3 gene) was cloned from Tichosporon penicillatum. Recently, a polygalacturonase-encoding gene from Saccharomyces cerevisiae has been cloned and overexpressed in several strains and the gene for an extracellular endopolygalacturonase from Kluyveromyces marxianus has also been described. Taking all the results together, the idea is now emerging that this type of yeast enzyme could offer an alternative to fungal enzymes for industrial applications.
To test the hypothesis that a switch in diet might cause changes in the abundance and composition of mucous-dwelling microorganisms, a short-term experiment was conducted with Atlantic salmon Salmo salar. Fish were fed on three different diets: pelleted S. salar feed, macroinvertebrates or pellets supplemented with an antibiotic. A fourth group of fish was deprived of food throughout the trial. Seven days after manipulating diets, significant differences were found in microbial density and community composition (quantified by different morphologically distinct colonies), particularly between fed and unfed animals. Moreover, food deprivation caused a rapid decrease in the number of epidermal mucous cells of the lateral skin, which may indicate a decrease in mucous secretion and explain differences in the diversity of mucous-dwelling microbiota observed in the fish. This is the first report of an effect of feeding regime on the abundance of microbial communities associated with cutaneous mucus of fishes.
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