Antarctic Yeast Research Articles

Synthesis and Characterization of an Exopolysaccharide by Antarctic Yeast Strain Cryptococcus laurentii AL100

An exopolysaccharide-producing Antarctic yeast strain was selected and identified as Cryptococcus laurentii AL₁₀₀. The physiological properties of the strain and its ability to utilize and biotransform different carbon sources (pentoses, hexoses, and oligosaccharides) into exopolysaccharide and biomass were investigated. Sucrose was chosen as a suitable and accessible carbon source. The biosynthetic capacity of the strain was studied in its dynamics at different sucrose concentrations (20, 30, 40, and 50 g/L) and temperatures (22 and 24 °C). The maximum biopolymer quantity of 6.4 g/L was obtained at 40 g/L of sucrose, 22 °C temperature and 96-h fermentation duration. The newly synthesized microbial carbohydrate was a heteropolysaccharide having the following monosaccharide composition: arabinose, 61.1%; mannose, 15.0%; glucose, 12.0%; galactose, 5.9%; and rhamnose, 2.8%. It was characterized by polydispersity of the polymer molecule, 60% of it having molecular mass of 4200 Da. The exopolysaccharide demonstrated good emulsifying and stabilizing properties with regard to oil/water emulsions and a pronounced synergistic effect with other hydrocolloids such as xanthan gum, guar gum, and alginate.

Effect of Different Factors on Biosynthesis of Exopolysaccharide from Antarctic Yeast

ABSTRACTPsychrophilic yeast Isolate 100, was selected as a producer of exopolysaccharide. A laboratory scheme was created for obtaining of the biopolymer. Biotechnological and physicochemical factors influencing its biosynthesis were investigated. It was determined that inocolum quantity from 6–10%, cultivated for 48 hours at 22° C, is suitable for maximum synthesis. The exopolysaccharide that was synthessed in the culture liquid was thermostable at 50°C to 70°C and it degrades at 80°C. The quantity of the sedimentary polymer, that was synthesis on environment with arabinose and mannose from Isolate 100, after incubation in ethanol at 4°C for 120 hours staying at the time of 120 hours in ethanol at 4 °C, was increased with 40%. The influence of the temperature over drying of the exopolysaccharide at 65°C and 105 °C was studied. At the low temperature of drying the polymer contained near 20% humidity. The effect of the experimental conditions (temperature, time, concentration of 2%, 3%, 4%, 5%) on the apparent viscosity values of the cultural liquid was studied. It was found to reveal a behaviour of non-Newtonian liquid. The power law equation of Oswald- de Waale describing the change of the apparent viscosity was derived.

Biomass Production by Antarctic Yeast Strains: An investigation on the Lipid Composition

ABSTRACTPsychrophilic yeast strains Rhodotorula glutinis AL107, Sporobolomyces roseus AL108, Cryptococcus albidus AL55, Cryptococcus laurentii AL56 and Cryptococcus laurentii AL58 isolated from soil sample taken from the region of the Bulgarien base on Livingston Island, Antarctica, were studied. The biomass production was followed after cultivation of the yeasts in a medium with pH 5.3 at 15°C for 120 h. The biomass concentration by psychrophilic yeast strains was: R. glutinis AL107-6.05 g/l, S. roseus AL108-5.78 g/l, Cr. albidus AL55, Cr. laurentii AL56 and Cr. laurentii AL58-6.52 g/l, 6.84 g/l and 6.24 g/l, respectively.The extracted and separated lipids from the samples were supplied to analysis and the compositions of fatty acids, phospholipids, sterols as well as tocopherols were determined. Unsaturated fatty acids, mainly oleic (58.6–63.5%) and of saturated palmitic (18.2–24.5%), predominated in triacylglycerols. Sterols (0.1–0.3%) were valued in the dry yeast biomass. The content of phospholipids, mainly phosphatidylcholine, phosphatidylinositole and phosphatidylethanolamine was found to be in the range of 0.2–1.6%. The quantity of tocopherols was 0–26.3 mg/kg. All of tocopherol classes were established.

Production of an exopolysaccharide by Antarctic yeast

Psychrophilic Antarctic yeasts produce polysaccharides in different concentrations. According to morphological, cultural, physiological and biochemical characteristics, the best producer strain was identified as Cryptococcus flavus A(51). The highest values for viscosity (59.1 mPa s) and crude polysaccharide productivity (5.75 g/L) were obtained in a medium containing 5 % sucrose and 0.25 % (NH(4))(2)SO(4), at 24 degrees C for 6 d. The chemical composition and sugar constituents of the crude exopolysaccharide were determined (92.5 % saccharides, 3.34 % protein, and 4.16 % ash). The monosaccharide composition of the exopolysaccharide obtained from C. flavus strain AL(51) was established (55.1 % mannose, 26.1 % glucose, 9.60 % xylose, 1.90 % galactose). The microbial biopolymer has a high molar mass and homogeneity: 82 % of it had M 1.01 MDa.

Phytase from antarctic yeast strain Cryptococcus laurentii AL27

Cryptococcus laurentii strain AL27 demonstrating significant potential for intracellular phytase production was selected by 2-step screening of Antarctic yeasts. The strain showed increased phytase activity in a culture medium with 40 g/L sucrose, KH2PO4 providing 5 mg/L phosphorus, and cultivation temperature of 24 degrees C, which relates it to psychrotrophic microorganisms. The enzyme kinetic characteristics according to sodium phytate were Km = 0.98 mmol/L, vlim = 33.3 micromol g(-1) min(-1). The enzyme had maximum activity at 40 degrees C and acted within a wide pH range: from 2.0 to 5.5, which is of positive significance for its direct inclusion into the feed of monogastric animals.

Production and Characterization of an Exopolysaccharide by Yeast

Antarctic yeast strains were investigated for exopolysaccharide biosynthesis and the Sporobolomyces salmonicolor AL1 strain was selected. It was studied for exopolysaccharide biosynthesis on different carbon and nitrogen sources. The investigations showed that sucrose and ammonium sulphate were suitable culture medium components for polymer biosynthesis. Exopolysaccharide formation by the yeast strain was accompanied by a decrease in the culture medium pH value from the initial pH 5.3 to pH 1.7–2.0. During the biosynthetic process, the dynamic viscosity of the culture broth increased to the maximum value of 15.37 mPas and the polysaccharide yield reached 5.63 g/l on a culture medium containing 5.00% sucrose and 0.25% ammonium sulphate at a temperature of 22 °C for 120 h. The crude polysaccharide obtained from Sp. salmonicolor AL1 featured high purity (90.16% of carbon content) and consisted of glucose (54.1%), mannose (42.6%) and fucose (3.3%). Pure mannan containing 98.6% of mannose was isolated from it.

A cold-adapted extracellular serine proteinase of the yeast Leucosporidium antarcticum.

An extracellular serine proteinase, lap2, from the psychrophilic antarctic yeast Leucosporidium antarcticum 171 was purified to homogeneity and characterized. The enzyme is a glycoprotein with a molecular mass of 34.4 kDa and an isoelectric point of pH 5.62. The proteinase is halotolerant, and its activity and stability are dependent neither on Ca(2+) nor on other metal ions. Lap2 is a true psychrophilic enzyme because of low optimal temperature (25 degrees C), poor thermal stability, relatively small values of free energy, enthalpy and entropy of activation, and high catalytic efficiency at 0-25 degrees C. The 35 N-terminal amino acid residues of lap2 have homology with subtilases of the proteinase K subfamily (clan SB, family S8, subfamily C). The proteinase lap2 is the first psychrophilic subtilase in this family.

Protein translocation across the endoplasmic reticulum membrane in cold-adapted organisms.

Secretory proteins enter the secretory pathway by translocation across the membrane of the endoplasmic reticulum (ER) via a channel formed primarily by the Sec61 protein. Protein translocation is highly temperature dependent in mesophilic organisms. We asked whether the protein translocation machinery of organisms from extremely cold habitats was adapted to function at low temperature and found that post-translational protein import into ER-derived microsomes from Antarctic yeast at low temperature was indeed more efficient than into mesophilic yeast microsomes. Analysis of the amino-acid sequences of the core component of the protein translocation channel, Sec61p, from Antarctic yeast species did not reveal amino-acid changes potentially adaptive for function in the cold, because the sequences were too divergent. We therefore analyzed Sec61alpha (vertebrate Sec61p) sequences and protein translocation into the ER of Antarctic and Arctic fishes and compared them to Sec61alpha and protein translocation into the ER of temperate-water fishes and mammals. Overall, Sec61alpha is highly conserved amongst these divergent taxa; a number of amino-acid changes specific to fishes are evident throughout the protein, and, in addition, changes specific to cold-water fishes cluster in the lumenal loop between transmembrane domains 7 and 8 of Sec61alpha, which is known to be important for protein translocation across the ER membrane. Secretory proteins translocated more efficiently into fish microsomes than into mammalian microsomes at 10 degrees C and 0 degrees C. The efficiency of protein translocation at 0 degrees C was highest for microsomes from a cold-water fish. Despite substantial differences in ER membrane lipid composition, ER membrane fluidity was identical in Antarctic fishes, mesophilic fishes and warm-blooded vertebrates, suggesting that membrane fluidity, although typically important for the function of the transmembrane proteins, is not limiting for protein translocation across the ER membrane in the cold. Collectively, our data suggest that the limited amino-acid changes in Sec61alpha from fishes may be functionally significant and represent adaptive changes that enhance channel function in the cold.

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for β-glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL2 and C. albidus AL3, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of β-glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl-β-D-glucopyranoside and salicin. The time course of growth and β-glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL2 and C. albidus AL3 synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties.

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l(-1) xylan and 10.2 g l(-1) yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml(-1) after 168-h shake culture at 4 degrees C. In addition, very little endoglucanase, beta-mannanase, beta-xylosidase, beta-glucosidase, alpha-L-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10 degrees C in a 5-1 fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0-5.5 and good stability at pH 4-9 (21 h at 4 degrees C). Although the enzyme was maximally active at 45 degrees - 50 degrees C, it appeared very thermolabile, showing a half-life of 78 min at 35 degrees C. At 40 degrees - 50 degrees C, it lost 71%-95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast.

Xylanase from the psychrophilic yeast Cryptococcus adeliae.

A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 +/- 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0 degrees-20 degrees C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

Structural characterization of a xylanase from psychrophilic yeast by mass spectrometry.

The complete structural characterization of the xylanase, a glycoprotein constituted of 338 amino acids, from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. The verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites and the investigation of glycosylation pattern were performed. This analysis revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures. The data obtained on both the N- and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophylic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophylic and thermophylic cells. The cysteine pairings were eventually identified as Cys173-Cys205 and Cys272-Cys278, with Cys89 showing a free thiol group. These data suggest that a common folding motif might occur within the entire xylanase family in which the second Cys is linked to the third one with the fourth and fifth joined together.

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.