Johnny Silva Cancer Research

Method 1 Microbial Production of Lactonic Sophorose Lipids

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1. Sterilize all media at 121°C and 1 bar overpressure for 20 min.

2. For maintaining C. bombicola ATCC 22214 in your laboratory, incubate the yeast strain on agar slants at 27°C for 48 h, and deposit at 4°C.

3. Transfer a loopful of cells into a 500-mL Erlenmeyer shake flask (with two inserts) containing 100 mL of seed culture medium; inoculate five flasks in parallel. After culturing for 36 h at 27°C on a rotary shaker (100 rpm), transfer 50-mL portions into 10 500-mL cultures placed in 2-L Erlenmeyer shake flasks (two inserts). Cultivate as mentioned above. Then combine these seed cultures, and transfer to the 50-L bioreactor filled with 45 L fermentation medium. Work at 250 rpm, 27°C, and at an aeration rate of 0.6 vol air/vol culture suspension and min. Do not adjust the initial pH value (5.2) until it reaches pH 3.5; now keep it constant with 5 M NaOH. After 36 h of cultivation time, start an additional feed of glucose (0.6 g/L) and oleic acid (0.4 g/L); finish feeding after 180 h.

4. Check the time-course of cultivation by using the following analytical methods:

a. Cell growth: Mix 10 mL of culture suspension vigorously with 10 mL ethanol/n-butanol = 1/1 (v/v). After centrifugation at 10,000g (20 min), dry the residual biomass at 105°C for 48 h and estimate the weight.

b. Total nitrogen: After centrifugation of 10 mL culture suspension at 10,000g- (20 min), take 0.2 mL supernatant for quantitative measurements according the to operating instructions of Lange's test kit Lato, LCK 338.

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c. Glucose: Buffer 4 mL of above supernatant (b) with 1 mL 1M KH2PO4/K2HPO4 (pH

7.0) solution, and drop 25 |L on a test strip (see operating instructions of Boehringer-Mannheim; Accutrend alpha).

d. Oleic acid: Extract 100 mL culture suspension twice with 100 mL ethyl acetate. Inject 1 |L of the extract (diluted with ethyl acetate to approx 2 g/L oleic acid) onto the GC column. The temperature profile should be as follows: 7 min at 165°C, 1 min 165^170° C, 7 min at 170°C, 5 min 170^220°C. Use oleic acid as standard.

e. Sophorose lipids: Use 1-100 |L of above ethyl acetate extract (d) for thin-layer chromatography (silica gel 60 plates). After development, spraying with detecting reagent and heating at 150°C for 1 min (pink/violet spots for glycolipids), take densitometer measurements according to the operational instructions of Desaga. As standard, use isolated sophorose lipid mixture. Rf values: 0.1-0.60.

5. Stop the cultivation after 200 h. After pumping the culture broth into a glass vessel followed by dilution (1/1) with water, the crude product separates at the bottom over night. Take off the aqueous phase (containing the yeast cells), and wash the precipitate with ice-cold water. Remove the aqueous phase again, and dry the drained product under reduced pressure.

6. To separate the individual sophorose lipids, load the column for medium pressure chromatography with 15 g crude products. Elute the components by 2% steps (900 mL/step) beginning with the highest chloroform/methanol ratio. Evaporate the 300 mL fractions to dryness, and detect with thin-layer chromatography.

7. Influence of crude glycolipids on the surface tension of water: Dissolve 100 mg in 10 mL dichloromethane. Add 10 vol of this stock solution, from 0.5 to 500 |L, to 20 mL distilled water placed in 10 special vessels. Add in each case pure solvent to reach a final volume of 500 |L throughout. After sonication (3 x 1min, T < 40°C, rank 4) with, e.g., a sonifier B 30 (Branson, Heusenstamm, GER), measure the surface tension of water at 25°C; use the Autotensiomat (ring method) according to the operational instructions of Lauda/Wobser GmbH & Co. KG.

Method 2: Microbial Production of Dodecyl-Sophorosides

1. Sterilize all media at 121°C and 1 bar overpressure for 20 min.

2. For maintaining the yeast strain, see Subheading 3.1., step 2.

3. Transfer a loopful of cells into 100 mL seed culture medium placed in a 500-mL Erlenmeyer shake flask (with two inserts). Incubate at 30°C on a rotary shaker (100 rpm) for 48-72 h. Add 10 mL of the seed culture to 500 mL of fermentation medium in a 2-L Erlenmeyer flask (with two inserts). Incubate under above conditions and, after 48, 72, and 96 h, add 8 g/L portions of 2-dodecanol to the microbial culture. Do not adjust the pH.

4. Check the time-course of cultivation by using the following analytical methods:

a. Cell growth.

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