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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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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)
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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.
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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).
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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
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Bioactive AgentsBioactive Agents
Septic shock antagonists
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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.
Industry/University Cooperative Research Center Industry/University Cooperative Research Center -- BiocatalysisBiocatalysis and and BioprocessingBioprocessing of Macromoleculesof Macromolecules
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).