Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

UNLABELLED: Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. PRACTICAL APPLICATION: Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes.

Characterization of the marine propionate-degrading, sulfate-reducing bacterium Desulfofaba fastidiosa sp. nov. and reclassification of Desulfomusa hansenii as Desulfofaba hansenii comb. nov.

A rod-shaped, slightly curved sulfate reducer, designated strain P2(T), was isolated from the sulfate-methane transition zone of a marine sediment. Cells were motile by means of a single polar flagellum. The strain reduced sulfate, thiosulfate and sulfite to sulfide and used propionate, lactate and 1-propanol as electron donors. Strain P2(T) also grew by fermentation of lactate. Propionate was oxidized incompletely to acetate and CO(2). The DNA G+C content was 48.8 mol%. Sequence analysis of the small-subunit rDNA and the dissimilatory sulfite reductase gene revealed that strain P2(T) was related to the genera Desulfonema, Desulfococcus, Desulfosarcina, 'Desulfobotulus', Desulfofaba, Desulfomusa and Desulfofrigus. These genera include incomplete as well as complete oxidizers of substrates. Strain P2(T) shared important morphological and physiological traits with Desulfofaba gelida and Desulfomusa hansenii, including the ability to oxidize propionate incompletely to acetate. The 16S rRNA gene similarities of P2(T) to Desulfofaba gelida and Desulfomusa hansenii were respectively 92.9 and 91.5 %. Combining phenotypic and genotypic traits, we propose strain P2(T) to be a member of the genus Desulfofaba. The name Desulfofaba fastidiosa sp. nov. (type strain P2(T)=DSM 15249(T)=ATCC BAA-815(T)) is proposed, reflecting the limited number of substrates consumed by the strain. In addition, the reclassification of Desulfomusa hansenii as a member of the genus Desulfofaba, Desulfofaba hansenii comb. nov., is proposed. A common line of descent and a number of shared phenotypic traits support this reclassification.

Assessment of yeast diversity in a marine environment in the south of Portugal by microsatellite-primed PCR.

The occurrence and diversity of yeasts in seawater was investigated in a study site located 20 Km off Faro, Portugal, above the Alvares Cabral Trench. A total of 43 water samples from different layers (above the permanent thermocline, under the thermocline and near the bottom) and directly from the surface, originated 234 isolates. All the isolates were identified using a molecular approach that included, in a first stage, MSP-PCR fingerprinting. A total of 31 MSP-PCR classes were formed, 8 for the pigmented yeasts and 23 for the non-pigmented yeasts. The pink coloured isolates were identified by direct comparison of the new fingerprints with those obtained for representative strains of the various species. For identification of the non-pigmented yeasts, a representative isolate of each MSP-PCR class was selected for sequence analysis and compared with reference sequences. The five most abundant yeast species were Sakaguchia dacryoidea, Pseudozyma aphidis, Rhodosporidium babjevae, R. diobovatum and Debaryomyces hansenii. The distribution of isolates and species in the major taxonomic groups indicated that the number of basidiomycetous yeasts and their diversity are prevalent in relation to their ascomycetous counterpart. Diversity indices were determined and superficial water and water near the bottom had the highest diversity. The sampling effort effectiveness was estimated, and found to correspond to approximately 60% of the species present. MSP-PCR identification proved suitable for pigmented basidiomycetous yeasts and, when used in conjunction with sequence analysis, was effective for the characterization of non-pigmented populations. Our results indicate that the MSP-PCR fingerprinting method is appropriate for the characterization of large groups of isolates due to its simplicity and good reproducibility.

Debaryomyces hansenii growth in nonsterile seawater ClO2-peptone-containing medium.

We found that the marine yeast Debaryomyces hansenii strain C-11 (CIBNOR yeast collection, La Paz, Mexico) is highly tolerant to chlorine dioxide (ClO2), a powerful biocide agent. A direct application of this observation is the fermentation of the yeast in a nonsterile medium with an initial concentration of 0.3 mg/L of ClO2. The disinfectant helps to avoid the growth of unwanted microorganisms while allowing the development of the yeast. Because the concentration of ClO2 decreases during the fermentation, we ascribe to D. hansenii cells a "biocontrol" action that contributes to the collection of a contaminant-free yeast cell biomass.

Copper-zinc superoxide dismutase from the marine yeast Debaryomyces hansenii.

We have isolated the cytosolic form of Cu-Zn superoxide dismutase (SOD) from the marine yeast Debaryomyces hansenii. This enzyme has a subunit mass of 18 kDa. The preparation was found to be heterogeneous by IF electrophoresis with two pI ranges: 5.14-4.0 and 1.6-1.8. The enzyme preparation had a remarkably strong stability at pH 6.0-7.0, surviving boiling for 10 min without losing more than 60% of activity. On Western blots, this enzyme was recognized by antibodies raised in rabbits against D. hansenii extracts, while only a weak cross-reaction could be detected using antibodies generated against either Saccharomyces cerevisiae or bovine erythrocyte Cu-Zn SODs. In sequencing analysis, a peptide obtained by trypsin digestion was found to have 85% identity to the S. cerevisiae Cu-Zn SOD.