Yeast exoglycoproteins produced under NaCl-stress conditions as efficient cryoprotective agents
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Yeast Exoglycoproteins Produced Under NaCl-Stress Conditions as Efficient Cryoprotective Agents E. Breierova
0 Culture Collection of Yeasts, Institute of Chemistry, Slovak Academy of Sciences , Dubravska cesta 9, 842 38 Bratislava , Slovakia
Six extracellular yeast glycoproteins were prepared from three yeast species in osmotic equilibrium and unequilibrium environments and used as non-penetrating cryoadditives. Glycoproteins secreted by the strain Dipodascus australiensis into growth medium containing NaCl (8% w/v) were found to be the most effective cryoadditives. It was possible to use these glycoproteins alone (without DMSO as penetrating agent) for the cryoprotection of the studied yeasts. Many factors that affect the successful freeze preservation of yeast cells have been documented (1). The crucial problem during preservation is to ensure genetic stability. The viability of the cells after preservation may correlate with the extent of protection against dehydration stress and with the transition of water to ice crystals (2,3). Survival and stability of all strains preserved in liquid nitrogen (196?C) is always higher in the presence of a mixture of penetrating and non-penetrating cryoprotective agents (4) than in the presence of non-penetrating cryoagents only (5). The basic feature of nonpenetrating cryoprotective agents is the ability of their hydrophilic groups to bind water, giving rise to a phenomenon often described as "bound water" (6). In higher cells, glycoproteins have various functions such as surface receptors, cell-cell mediators, components of the extracellular matrix, cryoprotectors, etc. The protein moiety of most glycoproteins is the functional part, while the carbohydrate moiety contributes to the attainment of an adequate tertiary structure and modifies the glycoprotein molecule making it more resistant to degradation, and facilitates its secretion (7). This paper describes the cryoprotective activity of six extracellular yeast glycoproteins prepared from three yeast species cultivated under two different conditions.
MATERIALS AND METHODS
The following psychrophilic species were used: Candida graminis CCY 29-133-1, Candida capsuligena
CCY 29-143-1, Leucosporidium scottii CCY 64-1-1, Phaffia rhodozyma CCY 77-1-1, Leucosporidium
antarcticum CCY 64-3-1, Candida diffluens CCY 29-132-1, Sympodiomyces parvus CCY 73-1-1,
Dioszegia hungarica CCY 18-1-1, Candida frigida CCY 29-144-1, Candida gelida CCY 29-121-1. All
yeast strains were obtained from the Culture Collection of Yeasts (Institute of Chemistry, SAS,
Basal cryoprotective medium
Basal cryomedium consisted of 7g wort extract, 0.3g yeast extract (Difco), 0.5g peptone (Difco) per 100 ml of tap water. The medium was three times sterilized by heating at 100 C for 1h on three separate days.
After sterilization of the basal cryomedium, DMSO (Fluka, Switzerland) was added as a penetrating
cryoagent to give a final concentration of 10% (v/v) in the first series of cryomedia. The second series
was prepared without DMSO. All cryomedia contained the crude ethanol precipitates of the different
extracellular yeast glycoproteins of three yeast species. These non-penetrating cryoagents were added
before sterilization into cryomedia to give a final concentration of 0.27%.
Cultivation conditions for preparation of the extracellular yeast glycoproteins
Control condition optimal mineral cultivation medium with 2% glucose contained per l: 4g yeast extract,
10g (NH4)2SO4, 1g KH2PO4, 0.2g K2HPO4, 0.1g NaCl, 0.1g CaCl2, 0.5g MgSO4, and 1 ml
microelements solution (1.25g H3BO3, 0.1g CuSO4, 0.25g KI, 1g MnSO4, 0.5g FeCl3, 0.5g
(NH4)6Mo7O24, 1g ZnSO4 contained per l). The optimal cultivation temperatures used were 28 C for
Dipodascus australiensis and 17 C for Waltomyces kononenkoe and W. lipofer (Table 1). The cells were
precultured aerobically in the test tubes to the stationary phase of growth and then cultivated on a
reciprocal shaker to the stationary phase in 1 l flasks with 500 ml of optimal mineral medium. After
centrifugation crude extracellular glycoproteins were isolated by precipitation from the supernatant by
adding two volumes of 96% (v/v) ethanol and subsequent centrifugation. The ethanol precipitate was
dissolved in distilled water, dialyzed against distilled water and freeze-dried.
To produce the stress conditions, 4% (W. kononenkoe, W. lipofer) and 8% NaCl (D. australiensis), was added to the control medium.
Preservation in liquid nitrogen
The cell suspension (0.1 ml, 107 - 108 cells in 1 ml) and cryoprotective medium (0.5 ml) were pipetted
into sterile 2-ml polyethylene ampoules (Koh-i-noor, Dalecin), and rapidly frozen in liquid nitrogen (no
later than 1h after pipetting). Three ampoules were used for each strain as well as cryomedium. Cultures
had been stored for one year. After 1 year of storage in liquid nitrogen, the cultures were thawed in a
37 C water bath for 30 min. opened, and recultivated.
Survival of the storage culture
RESULTS AND DISCUSSION
The six crude glycoproteins were tested to determine their effect on the survival of the 10 psychrophilic yeast strains stored in liquid nitrogen.
The strains could be divided into three groups on the basis of their survival rates in the basal medium containing DMSO (8) (Figs. 1, 2). The strains survived only to a limited extent (to 4%) or did not survive in the basal medium without any cryoadditive.
Group I comprised strains with the highest survival rates (20 ? 6%). These strains were typical
psychrophilic species. Each of them produced some protective agent such as carotenoid pigment or
slime. The most suitable glycoprotein cryoprotectants were G2, G4 and G6 produced under
NaClinduced stress conditions. Without DMSO (Figs. 1, 2) the most effective cryomedium was the
one containin2g. TGhe average survival rate was about 75%.
Group II comprised four strains with lower survival rates (about 15 ? 5%). The strains of D. hungarica and C. gelida survived somewhat, while C. graminis and L. scottii survived to the lesser extent.
Group III comprised the most cryosensitive strains (survival rates 0 - 5%).
In the DMSO-free cryomedium only small differences in the percentage of survival when using various
glycoproteins were observed. The glycoproteins produced by the strains of D. australiensis under stress
conditions (8% NaCl in the cultivation medium) had the highest influence on the cryosensitive
psychrophilic yeast species (Figs. 1, 2). The glycoprotein G2 was the most effective for all strains.
The order of the effectivity of glycoproteins wa2s>:GG1 > G6 > G5 > G3 > G4.
The extracellular yeast glycoproteins produced by the genus Waltomyces (G3, G4, G5 and G6) were not
very effective as cryoprotective agents. The ability of these cultures to grow at the higher concentration
of NaCl is probably dependent on the structure of the glycoproteins produced. During freezing, the
residual solution becomes hypertonic with regard to the intracellular compartment, and thus the cells are
subjected to a fast decrease of osmotic pressure. The glycoproteins isolated from the 8% NaCl medium
were efficient as cryoprotective agents.
The results of the study of the survival rates will be used to develop experimental protocols that minimize osmotic stress without the penetrating cryoprotectant DMSO. The application of the above results to another type of eucaryotic cells has also been described (9).
Group I (with DMSO):
Group II (with DMSO):
Ph. rhodozyma L. antarcticum
Group II (without DMSO):
Group III (with DMSO): Group III (without DMSO):
The author gratefully acknowledges the support from VEGA grant number 2/4145.
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