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Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules

What are Sophorolipids?What are Sophorolipids?Yeast derived Yeast derived glycolipidsglycolipids, consist of , consist of dimericdimeric sophorosesophorose linked to linked to longlong--chain hydroxy fatty acidschain hydroxy fatty acids, produced by , produced by Candida Candida speciesspecies(e.g. (e.g. C. C. bombicolabombicola ATCC 22214)ATCC 22214) when grown on carbohydrates, when grown on carbohydrates, hydrocarbons, vegetable oils or their mixtures. Produced as a hydrocarbons, vegetable oils or their mixtures. Produced as a mixture of six to eight differentmixture of six to eight different hydrophobic sophorosides.hydrophobic sophorosides.

O

OHH O

O CH

CH 3

H O

OH

OH

O O

(CH 2)6

CH

CH

(CH 2)7

COOH

R 2O

R 1O

S1. R1 = R2 =Ac S5. R 1 = R 2 = AcS2. R1 = H ; R 2 = Ac S6. R 1 = H; R 2 = AcS3. R1 = Ac; R 2 = H S7. R 1 = Ac; R2 = HS4. R1 = R 2 = H S8. R 1 = R 2 = H

O

O

CO

O

OHH O

OH

OH

O CH

CH 3

CH

CH

O

(CH 2)7

(CH 2)6

R1O

R 2O

Lactonic form Acidic form


SL production with linear increase in agitation at different SL production with linear increase in agitation at different volumetric levelsvolumetric levels -- Existence of an optimal aeration windowExistence of an optimal aeration window

An oxygen transfer An oxygen transfer rate (OTR) rate (OTR)

between 50 and between 50 and 80 80 mMmM OO22/L h/L h--11

gave the gave the maximum maximum volumetric volumetric

product product formation (1formation (1--1.5 1.5 g/L hg/L h--11) and SL ) and SL volumetric yield volumetric yield

(350 g/L)(350 g/L)

(Model studies with Oleic acid)

Culture Time (day)

0 2 4 6 8 10 12 14 16

Spec

ific

Prod

uctio

n,

g SL

/g b

iom

ass

per d

ay

0

1

2

Agi

tatio

n Sp

eed

(rpm

)50

100

150

200

250

300

350

400

30 mL cultures70 mL cultures110 mL cultures150 mL cultures agitation


Oxygen Transfer Rate (OTR) (mM O2/L h-1)

0 20 40 60 80 100 120

SL p

rodu

ctio

n (g

/L d

ay)

0

10

20

30

40

50

60

SL production depends on the aeration conditions.SL production depends on the aeration conditions.There exists an optimal range of OTR in flasksThere exists an optimal range of OTR in flasks.

V Guilmanov, A Ballistreri, G Impallomeni, R.A. Gross, “Oxygen Transfer Rate and SophoroseLipid Production by Candida bombicola”, Biotechnol. and Bioeng. 77(5), 489-494 (2002)


OO

O

HO

HO

HO

O CHCH3

(CH2)6

CHCH

(CH2)7

COOR

HO

OH

OH

OH

Simple Derivatives of Natural Products

Sophorolipid mixture

OO

O

HO

HO

O CHCH3

(CH2)6CHCH(CH2)7

CO HO

OH

OH

OH

O

OO

O

HO

H3COO

H3COO

O CHCH3

(CH2)6

CHCH

(CH2)7

COOR

HO

OH

OH

OH

NaOR

R = Me, Et, Bu and Hex

Bisht, Gross.., J. Org. Chem., 1999, 64, 780Singh, S. K, Gross…J. Org. Chem.; 68(14); 5466 (2003)

Novo 435

Vinyl acetate,Novo 435

Also Monoacylation


Surface Tension of Esters of SophorolipidSurface Tension of Esters of Sophorolipid

30

40

50

60

70

0.1 1 10 100 1000 2000

Concentration, 10 -6M

Sophorolipid methyl ester

Sophorolipid ethyl ester

Sophorolipid propyl ester

Sophorolipid butyl ester

Sophorolipid hexyl ester

T= 45 0C

In collaboration with: Prof. P. SomasundaranColumbia University


Critical micelle concentration of sophorolipid esters as a function of alkyl chain length in comparison to alkyl glucosides and alkyl maltosides

1x10-6

1x10-5

1x10-4

1x10-3

1x10-2

1x10-1

6 8 10 12 14 16 18 20 22 24 26Carbon Number

alkyl glucoside

alkyl maltoside

sophorolipid esters

Zhang, L., Somasundaran, P., Singh, S. K., Felse, A. P., Gross, R.A. Synthesis and interfacial properties of sophorolipid derivatives Colloids and Surfaces A: Physico-chem. Eng. Aspects 240 (2004) 75-82


Self-assembly of surfactants and block copolymers

Macromolecules, 1998, 31, 5300-5308; 7746-7755; 1999, 32, 5836-5845; 6326-6332. J. Polym. Sci., Polym. Phys., 1998, 36, 889-900.

In waterScience, 2001, 291, 1944-1947


In collaboration with: Prof. Shuiqin Zhou --CUNY College of Staten Island

Self-Assembling of Sophorolipids

O

OH

OH

O

OR1

OC H 3

HO

O

O

R2O

OHO

OH

O

O H

O H

O O

O

R 2 O O H

O

O H O

C H 3

O R 1

R1 and R2 =COCH3R1 and R2 = HR1=COCH3, R2 = HR1 = H, R2 = COCH3

Sophorolipids

Free acidic SLHydrophobic

Hydrophilic

O

OHO

HOO

OH

CH3

COHO

HO OH

OHO OH

pH


Self-Assembling of Sophorolipids

Big helical ribbons (e.g., ~11 µm wide and hundreds µm long)Zhou, S., Xu, Chang.., Wang, J., Gao, W., Akhverdiyeva., Shah, V., Gross, R. A. Supramolecular Assembles of a Naturally Derived Sopholipid. Langmuir 2004, 20, 7926-7932

Free acidic SL dissolved in dilute HCl, final pH = 4.1, C = 2.2 mg/mL50 µm50 µm


Molecular modeling

of SL-COOHmolecules (A)

and a possibleinterdigitated

lamellar packing model of SL-COOH moleculesin the giantribbons (C).

B: interdigitated dimerA: SL-COOH

C: Long-range interdigitated packing


Reported Effects of SophorolipidsSeemingly well tolerated in mice and rats without conflicting reports.No toxicological effect of non-modified or native sophorolipids in rats with 208 mg/kg/dayoral LD 50 (rates + mice): 10-16 g/kg; mice: 5.8 to 6.6 g/kg. The oral LD of SL is estimated at 12.5 g/kg.

Ikeda Y. et al. J. of Toxicological Sciences, 11 (3): 197-211 and 213-224, August 1986.

No measurable conseqeuence with 12.5 g/kg SL in rates or 6 g/kg in mice.

Otto R. T. et al. Appl. Microbiol. Biotechnol (1999) 52: 495-501.


OO

O

HO

HO

HO

O CHCH3

(CH2)6

CHCH

(CH2)7

COOR

HO

OH

OH

OH

Previous work done by our group

Sophorolipid mixture

OO

O

HO

HO

O CHCH3

(CH2)6CHCH(CH2)7

CO HO

OH

OH

OH

O

OO

O

HO

H3COO

H3COO

O CHCH3

(CH2)6

CHCH

(CH2)7

COOR

HO

OH

OH

OH

NaOR

R = Me, Et, Bu and Hex

Bisht, Gross and Kaplan, J. Org. Chem., 1999, 64, 780.

Novo 435

Vinyl acetate,Novo 435

Also Monoacylation


Copolymerization of Monoacryl Sophorolactone with Acrylamide

,

m

H 2N C O C H = C H 2

AIBN

H C C H 2C H 2H CC O N H 2 C O

O

n

70 o CO H

O

O

O H

O

C H 3O O

O H

O H

O

O C O C H = C H 2

O H

DMF

SL

8 h

Bisht, Gao, Gross, Macromolecules, (2000).


5.000.060.020.0075.001.67Candida tropicalis5.000.060.060.025.001.67Candida antartica5.001.675.005.005.001.67Candida albicans

5.000.060.020.0075.001.67Streptococcus agolatiae

5.000.560.060.06N/A1.67Bacillus subtilis

5.001.675.005.005.005.00Salmonella cholerasesuis

5.005.005.000.0070.56> 5.00Rhodoccocuserythropolis

5.005.005.005.005.05.00Ralstonia eurtopha5.000.060.060.025.001.67Moraxella sp.5.001.675.005.005.001.67Escherichia coli

(mg/ml)(mg/ml)(mg/ml)(mg/ml)(mg/ml)(mg/ml)

Natural SL

SL Methyl Ester

SL DiAcetate Ethyl Ester

SL Monoacetate Ethyl

Ester

SL-AcidHexyl SLMicroorganisms

Shah V, Doncel GF, Seyoum T, Eaton KM, Zalenskaya I, Hagver R, Azim A, Gross R. Sophorolipids, microbial glycolipids with anti-human immunodeficiency virus and sperm-immobilizingactivities. Antimicrob Agents Chemother 49:1-8 (2005)

Antimicrobial Activity


Bioactive AgentsBioactive Agents

Septic shock antagonists


CecalCecal LigationLigation to induce sepsisto induce sepsis

All animals were anesthetized with an intraperitoneal injection of Nembutal (40mg/kg).The cecum was ligated just below the ileocecal valve with a 3-0 silk ligature. The antimesentric cecal surface was punctured with a 16-gauge needle proximal to the ligature. The abdominal incision was then closed in two layers.The animals were randomly assigned to one of two groups (n=25 per group).To determine the efficacy of intravenous administration of Sophrolipids, animals were randomly assigned to two additional groups (n=25 per group).Control animals received physiological saline, while experimental animals received sophrolipids (5 mg/kg) intravenously (IV) via tail vein injection at the end of the operation. The animals were then monitored over 36 hours and survival rate was compared between the experimental and control groups. The endpoint of the study was lethality

To test the benefits of Sophrolipids in an in vivo system model of sepsis, 100 Male Adult Sprague-

Dawley rats (200-240g) were used.


Intravenous administration:The survival rate at 36 hours was significantly increased by 34% by IV injection of SL (81.8% survival rate) compared to Placebo controlled group (47.8% survival rate) (P<0.05).

Bluth MH, Kandil E, Mueller CM, Shah V, Lin YY, Zhang H, Dresner L, Lempert L, Nowakowski M, Gross R, Schulze R, Zenilman ME. Sophorolipids block lethal effects of septic shock in rats using a cecalligation and puncture model of experimental sepsis. Crit Care Med Vol. 34, No. 1 (2006).

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