1156

OLEOCHEMICALS

Biosurfactants face increasing interest

..... WjlidHI' ", -ell-tHz-COOK,leH,It,'",.,

RIID....a,id 113, "'·Clt -eHz-COOH,ICHzl,,'"RftO.. O\I~ A~' 11'11

There are a great number of natu-rally occurring surfacrams ofanimal, plant and microbial ori-

gins. Soaps, rosin, shellac, saponin.lecithin and gums have been utilizedas surfactants and detergents. Thedominance of petrochemical-derivedsurfactants may be passing. Thereforenatural surfacrants are being evaluatedfrom the aspect of the importance ofenvironmental protection, safety andmildness. With this increasing etten-lion, natural surtactams were recentlyrenamed as biosurtaciems. and theavailability of biosurfactants has beenclarified (1-9), Microbial and othernaturally occurring surfacrants are sur-veyed here and are proposed as novelsurfectanrs. Typical biosurfuctants arereviewed with regard to their occur-rence. chemical structure. yield andproperties. as well as findings onfunctionality and applications. focus-ing mainly on our results.

GlycolipidsRhamnolipids. Jarvis and Johnson(J 0) obtained rhamnolipid R 1 and R3in the yield of 2.5 gIL from a culturebroth of Pseudomonas aeruginosa anddiscovered their antibiotic properties.Hauser and Karnovsky (II) andEdwards and Hayashi (12) invesrigar-ed their production and characterize-lion. Later. rhamnolipid R I and R3were studied again. as the microbe-secreting products of petroleum-degrading microorganisms (12-15). Itwas already known that Pseudomonasspecies produce four kinds of mamno-lipid homologues from hydrocarbonsas the sole carbon source. These con-sist of one or two units of rhamnoseand a carboxylic moiety as the

SCheme A

TIWonide __ P'''fIIITOdftw INRlRM by _

I~ oftlwNtIIiDIoaIl_.ofMilluUW andC_R ......Io,Agacyofl_lkinruandT«IuwIogy. Mm.I·I.HigfuIri. T>vbIbtJ.city.''''''''gi.305. /aptm.

hydrophilic portions and 24hydroxy4decanoyl-z-hydroxydecanoatc. withand without decenonte as thehydrophobic moiety. Yamaguchi et al.(16) found decenoate-bearing rharnno-lipid homologues of RA and RB (14gIL; RA:RB = I:3) in a study of theproduction of single-eel! protein.These rhamnolipid homologuesshowed strong surface activities(17.18). antibacterial and antiviralactivities (15) and enhancement of thegrowth rate (19.20) and spreading (21)of microorganisms (Scheme A).

Itoh et al. (15) obtained R I: 3.7gIL and R3: 3.5 gIL from P. aerugi-nosa Ky 4025. Syldatk et 01. (17.22)succeeded in increasing the yield from12.8 gIL using growing cells of Pseu-domonas DSM 2874 to a 2.2-foldyield using resting cells. Furthermore.they also prepared two new types ofrhamnolipids. R2 and R4. using theresting cells. Koch et al. (23) accom-plished rhamnolipid production in lac-tose-based media or whey as the solecarbon sources by a genetic mutant ofP. aeruginosa, which was inserted intoEscherichia coli genes. Rhnmnolipidproduction from olive oil as the car-bon source was investigated aiming atthe utilization of nonhydrocarbon sub-

strate (24). P.aervginosa UG Iisolated from oil-contaminatedsoil samples wasfound to excreterhamnolipids.etc. (25).

Trehaloselipids. Trehaloseoften has beenfound in insectslind rnicroorgan-

isms, and is considered to be one ofthe storage carbohydrates. Trehalosedimycolate has been well known asthe cord factor that assists the cord-like growth of mycobacteria (26,27).The cord factor shows toxic activityas well as useful immunosrimulantand tumor inhibition activity (28).Then. trehalose lipids were foundagain by Suzuki et at, (29) from theculture broth of Arthrobacter parof:[inius KY 4303 grown on n-paraffinin a yield of 0.5 giL Wagner et 01.(30-32) and Batrakov and co-workers(33) also found trehalose Lipids fromthe culture broth of Rhodococus ery-thropolis and Mycobacterium paraf-finicum. Rapp et al, (31) tried to usethe trehalose lipid-containing brothfor the tertiary recovery of petroleumin Germany (Scheme B).

CHrO~ ~.10;-\ OK

R~O OKOR CHzOH

SchemeB

R. erythropolis also produced sue-cinoyl trehalose terraesrers in a yieldof O. I gIL with trehalose corynomyco-late under the limitation of nitrogensources in (he culture broth (34).Using the resting cells Rapp (31) et at .increased the yield up to 7.9 gIL. Onthe other hand. Uchida et al. (34-36)isolated new types of succinoyl tre-halose lipids in a yield of 15 gIL asthe result of screening of the hydro-carbon-assimilable microorganism. R.erythropotis SD474. in a higher alka-line condition. These compounds weresurface-active. especially, as disper-sants and as emulsifiers on the basisof the rnultifuncrionaliry of their mul-

INFORM. Vol. 4. no. 10 (October 1993)

1157

Corynebacterium diphtheriae andMycobacterillm smegmatis, from glu-case as the carbon source: four molesof 3-hydroxydecanoic acid ester (60)

tiple hydrophobic and hydrophilicportions (37). Furthermore, Kawai etat. (38) reported antiviral activity ofthese compounds.

Sophorolipids. Sophorose isknown as the molecular constituent ofstevioside. Spencer and co-workers(39,40) found sophorolipids as theextracellular oil of Toturopsis bambi-cola (magnoliae) in the culture brothusing glucose as the carbon source.The yield was 0.5 gIL (41), and a sub-stitution for musk perfume was tried.Sophorolipids consist of more thannine kinds of homologues as well asboth the free acid and lactone formsof the terminal carboxylic moiety.Inoue et al.(42) and Inoue and Itoh(43) achieved a high yield ofsophorolipids (120 gIL) from vea-etable oil and glucose as the carbonsource. which were subsequentlyderived to the propyleneglycoladducrs for cosmetic applications(44). There has been much substantialresearch (45-48) 10 improve the yieldby convening substrates from saf-flower, sunflower. palm and canol aoils, and oleic acid, etc. with glucose.The highest yield reported is 148 gILfrom canola oil by Zhou et 01. (48).

Others. Ustilagic acids (49-51):Ustilago maydis. yield: 3-4.5 gIL fromglucose as the carbon source. antibac-terial activities.

Mannosylerythritol lipids (52-55):Candida sp. S-IO, 8-7 yield: 40 gILfrom hexadecane.

Sucrose lipids: 6-0-corynomy-coloyl sucrose and dicorynomylcoloylsucrose (56): A. paroffimus, total yieldof 0.5 gIL from sucrose; carbohydratefauy acid esters (57): lipase, yield wasmore than 60% from sucrose or glu-cose and long chain fatty acid as thesubstrates; corynomycolates of carbo-hydrates such as sucrose, maltose,mannose and maltitol, etc. (58): rest-ing cells of Arthrobacter sp. DSM2567, grown on glucose at the firststage according to the biotransforma-tion technique.

Regioselective sucrose-based sur-factants seem 10be the most importantcarbohydrate esters as fine chemicalsformed from sucrose, and they can bemass produced.

Glucose lipids: 6-0 -corynomy-coloyl glucose (59): cell bodies of

1""'"~O-O-tll

CHI CHi~O-O-{II 1¢1il1$~lIr ~III eH,

~

CO-O-CH (CH,I!HO ell, ell, CHI

O-fll ~III1$Oil ~H, CHI

(ell,I,

/"SchemeC

(Scheme C).Alkyl glucoside (61): ~-glucosi-

dase from Trichoderma virdie, yield:2%.

Alkyl xylosides (62): [l-xylosidasefrom Aspergillus niger. yield: 100%.

Fructose lipids (56.63): 6-0-corynomycoloyl fructose and 1.6-dicorynomylcoloyl fructose, and high-er order esters of fructose.

Tetraglucose lipids (64.65):Micropolyspora NS-J 085K. yield:22.5 gIL from sucrose.

Pentasaccharide lipids (66) Nocar-dia corynebecrercldes, yield: 2.75 gILfrom n-Ct4-15 alkanes.

Substituted fatty acidsCorynomycotic acids (2-alkyl-3-hydroxy Jatty acids). Constituent long-chain fatty acids of trehalose esters arez-atkyt-S-hydroxy fatty acids.Corynebacteria produce corynomy-colic acid homologues ranging fromY8-36; Nocardia produce nocardorny-colic acids ranging from C50-56; andMycobacteria produce nocardomycol-ic acids more than Coo (27). Freecorynomycolic acids also were found(67.68). The chainlength of the hydro-carbon substrate influences the alkylchainlength produced by microorgan-isms (69) Corynomycolic acidsshowed surface activities in aqueoussolutions (68). However, the detailswere not clear because of the hetero-geneity of the alkyl chain lengths.Therefore, Ishigami and co-workers(70) prepared chemically the respec-tive homologues of corynomycolicacids as a kind of biomlmeric surfac-

I

R,·CH-CH-COOII, ,Oil RI

A"CUi_CnA,'e! -C'l

SchemeD

tants (Scheme D).Spicutisporic acid. Tabuchi et al.

(71,72) found a high yield production(110 gIL) of spiculisporic acid {(4S.55)·4 ,5 -d icarbox y-4- pen tadec an 0-!ide] using Penicillium spicnlisporumLehman 10-1 from glucose as thecarbon source under acidic incuba-tion in the study of succinic fermen-tation. This compound is the dehy-drated product formed in acidic con-ditions from 3-hydroxy-l ,3,4·telrade-canetricarboxylic acid, which is pro-duced enzymatically from lauric acidand 2-ketoglutaric acid of TeAcycle. Furthermore, it must beemphasized that the chemical struc-ture is exclusively the open-ring formand contains only the pentedecanealkyl chain in comparison with a dis-tribution of alkyl chainlengths inordinary surtacranrs.

The multifunctionality of spi-cutlsporic acid, as well as its safetyand biodegradability, seems to be use-ful for applications in the field of sur-factants and synthetic intermediates.

Others. Agaricic acid (3-hydroxy-1,3,4-tetradecanetricarboxylic acid),Polyporus officinatis Fris. This com-pound is known as an antisweat phar-maceutical.

c.o-Dtcarboxyhc acid (73): COl/-

dido cloacae MR-12, produced 1,14-tetradecanedicarboxyLic acid (55 gIL)from hexadecane as the carbon source.This product has the potential for useas an intermediate for musk perfumeand in engineering plastics. When theodd-number fatty acids are used assubstrates, the resulting dicarboxylicacids also comprise the correspondingodd-number carbons; for example,1,13-tridecane dicarboxylic acid isproduced from n-pentadecane as theoxidized product.

Hydroxy fatty acids (24,74): mono-and dihydroxyl-subsrituted fatty acidsare found as constituents of liposaccha-ride-type biosurfactants in organisms.

INFORM. Vol. 4, nO.10 (October 1993)

1158

GlyceridesPhospholipids. Soybean oil lecithin. amixture of phospholipids. has beenutilized commercially as a food emul-

sifier. Microbial phos-CH, pholipid production is..> CH!CIlzt.CHCK:,CO-. - CII-. -L.a-D -lU-L -~DI-L-ASp-O-Lt. -L -lu no I a dvan I a geo u sCH, I 0 I

because smaller yieldsare obtained than yieldsfrom plant origins. Thehydrogenized and lyse-lecithins recently havebeen developed for cos-metics and foods. Fur-

Lipo amino acids, JipopeptidesN-acy/ amino acids. Naturally occur-ring L-Tryptophan and L-phenylalaninehave weak hydrophobicity. The fonneris able to reduce the surface tension ofan aqueous solution to 54 mN/m (75).Both are hydrotropic agents (76). It wasreported thai P. rubescens producedornithine lipids (77) and Gluconobactercerinus produced cerilipin lipids (78).N-acyl hydroxy proline-taurine and N-hydroxy proline-cysteinic acid weredetected in the gastric juice of the Crus-tacea (79) (Scheme E).

IIHI-CHI- [CHIlI -CMIIH(CO-CMrCHIOCOJlII- JI,)

R," 3-1oy6",IIO""lIakR,"lan,. acid........

Cyclic Iipopeptiaes. Polymyxin Bis an amphipathic antibiotic which isproduced by Bacillus polymyxa (80).This is a cationic surfactant and caus-es disorder of lipid bilayers as well assolubilization of membrane proteins(5). Surfactin (81) (Subtilisin (82)] isan effective anionic surfactant consist-ing of a beptapeptide containing 3-hydroxy-I,3-methyl terradecancicacid. The yield is 0.8 gIL (5). Thisshows lysis for erythrocytes, bacteria.and blood fibrin clots, inhibition ofthe enzymatic activity of cyclic AMPphosphodiesterase. and inhibition ofheat denaturation of bovine serumalbumin (83). The cyclopepnde ringforms the B-sheet structure. Therehave been some other surfactin ana-logues consisting of different aminoacid compositions from surfactin(84,85). Matsuyama (84) found thatthe spreading growth of Serratiamorcescens, etc., depended on theexcretion of biowetting agents such ascyclo peptide. This also means thatmicroorganisms without flagellaacquire moving ability in the fractal-like growth of the microbial colonies(Schemes F and G).

................. G

BiopolymersEmulsan. Rosenberg er at. (86) report-ed the protein associated anionic Jipo-heteropolysaccharides having molecu-lar weights of about one million thatconsist of N-acetyl-galactosamine.uronic acid and N-acetyl amino sugaras the backbone chain and long-chainfauy and hydroxy-Iauy acids as thehydrophobic moieties. produced byAcinetobacter calcoaceticus (4-6).One emulsan homologue. o-emulsan.is dispersable.in water and gives lowviscosity. 5 cp at 1% dispersion.Emulsan shows an emulsifying actioneven in low concentrations(0.001-0.01%) and stabilizes its emul-sions. It has been investigated formany applications, such as a detergentfor oil tanks. spilled oil treatments. aviscosity reducer of heavy oil. and forseparation of heavy metals and cation-ic dyes (3) (Scheme H).

thermore there is interest in thesephospholipid homologues for use innew applications for liposcmes andoil-in-water lecithin emulsions (lipidmicrospheres) especially for medicalpurposes. In addition, both phospho-lipids and proteins are active compo-nents of lung surfactants mat are nec-essary for human respiration. It is alsonotable that diacyl phosphatidyl-ethanolamine, a kind of phospholipidhomologue. is one of the most effec-tive natural surfactants with an inter-facial tension of less than I mN/m forbexadecane. and a critical micelleconcentration (CMC) of 30 mg/L (90)(Scheme J).

,•"''''.,

H~H ~

"""roo'"........ ,

H-J3A gl)'ceroglycolipid. Finnertyand Singer (91) isolated an extracellu-lar trehalose-containing glycolipopep-tide produced by Rhodococcus sp. H-13A grown on hexadecane as me car-bon source. Fatty acyl groups arelinked to hydroxyl groups of theoligosaccharide-peptide backbonewith glycerol. The yield was increased(40.3 gIL) using a new techniqueinvolving the construction of a E.cali-Rbodococcus shuttle vector andplasmid transformation in Rhodococ-cus (92). The interfacial tensionreached extremely low values. forexample. 0.02 mN/m (1.8 mg/mLsolution) for decane at 40°C and0.00006 mN/m for undecane in thepresence of 0.5% pentanol as a cosur-factan!. This biosurfactant was a spe-cific emulsifier causing viscosityreduction. Oil displacement experi-ments from oil-bearing cores werecarried out successfully (Scheme J).

CHzOOCRDlGLVCERIOE 'lJI£lW..OS{ -GlUCOSE -GUICONC K.ID

....... J

........ H

Others. There have been someother microbial biosurfactants consist-ing of polymers composed of protein-lipid-polysaccharide (87). liposaccha-ride (88). mannan-fatty acid complex(88) and Iiposan (89). Their constitu-tional sequences generally were nOIdefined because of analytical difficul-lies.

INFORM. Vol. 4. no. 10 (October 1993)

Glycosyl glyceride. Oiglycosyldiglycerides (69): u-digtucosyt-, B-diglucosyl-, dimannosyl-, dtgalacto-syl- and galaclosylglucosyl digtyc-erides in microorganisms and plants;digalactosyl diglyceride, R. aummio-cus from hydrocarbons.

OthersNatural amphiphi/es. There havebeen a variety of natural amphiphileshistorically. such as gums, rosin.saponin, egg york lecithin, shellac,casein, bile salts, amphiphile ofsoapfish and so on. The recent trendtoward use of natural products hasincreased annually compared tochemicals from petroleum. Naturalpolysaccharides (gums) of plant andmicrobial origins have been devel-oped as food emulsifiers, stabilizersfor emulsion and dispersion systems,thickeners, gelling agents, coatingsor film-foaming materials and adhe-sives. Arabia gum, tragacanth gum,and argic acid are well known.Microbial polysaccharides such asxanthan gum, pull ulan, gellan gum,curdlan, and elsinan, and plantpolysaccharides such as guar gum,locust bean gum and carrageenanhave appeared successively in themarket. Hydrol yzates of wheatgluten and soybean protein also areused for similar purposes. Milk fatglobule membrane from whey isused as an emulsifier and a drugdelivery system (DDS). Humins arealso amphiphiles. Some saponinssuch as glychrrhizin. stevia. soya androot saponins have been commercial-ized as amphiphiles. sweeteners,food emulsifiers and for pharmaceu-tical purposes.

Chemical modifiers of biologicalsubstances. Chemical modification ofbiomarerials and biologically activesubstances are necessary to preparenew trpes of functional surfactantsand to "develop additional functionsneeded for particular uses.

Examples of the former are N-acylamino acids (glutamate, sarcosinate,alan ate. arginate, lysin ate), N-acylamino acid ethylene oxide (EO)adducts, C-alkyl amino acids, mono-glyceride esters of N-acetyl asperagl-nate, acylpeptides from collagenhydrolyzates, polyoxyethylene sor-

bitol oleates, dextrin palmitates, matli-101 monopelmhates. lanolin fatty acidesters and lanolin alcohol EO adducts.

Examples of the latter include thesurfactant structures of thiamine, pyri-doxine, brasticidin S (agriculturalpharmaceutical), methionine, quinine(sun-screen agent), diphenhydramine(antihistamine) and procaine in alkyl-sulfate salts. There are also lanolinfatty acid esters. lanolin alcohol EOadducts, rosin acid glyceride, rosinacid pentaerythritolester, polycxyethy-lene tocopherol and calciferol, pro-caine EO adduce, thiamine butyrate,pyridoxine Iany acid ester, testos-terone asparaginate and behenate ofcytosinearabinoside.

CharacterlsticsAs described for each of the com-pounds discussed in the previous sec-lion. the chemical structures of micro-bially produced biosurfactants aregenerally more complex and macro-molecular compared with convention-al synthetic surfactants. Accordingly,their molecules are comparativelybulkier than those of petrochemical-based surfactants. A comparison ofthe typical bulkiness of rhamoolipid Bwith sodium dodecylsulfate is shownin Figure I. These characteristicchemical structures of biosurfactantsare thought to be quite closely relatedwith their chemical and physicochem-ical propenies.

Biosurfactants exhibit excellentsurface activities, such as surface andinterfacial tension lowering(18.81,93), wetting or penetratingactions (84,93), dispersing and emul-sifying actions (37), detergency (94),gelling (95) and vesicle formation(95.96), foaming (19), demulsifying(I). flocculating actions (97). micro-bial growth enhancement (7,84,98).metal sequestration (5,99) and antimi-crobial (38) action. despite their bulkymolecular structures in comparison totraditional petrochemical surtacranrs.In addition. there are other importantcharacteristics of biosurfactants.

Biosurfactants' characteristics areattributed to their chemical structure.Namely. there are hydroxy fatty acids.steroids and isoprenoids as thehydrophobic portions, while saccha-rides (trehalose, glucosamine,

polysaccharides). TCA acids (z-keto-glutaric acid, citric acid), amino acids,cyclic peptides and glycerol are thehydrophilic portions. These are main-ly liposaccharide-type biosurfactantsas previously discussed.

Bulky biosurfactant molecules pro-vide several portioos capable ofexpressing functional control. depend-ing on the environment. The occupiedareas per molecule of biosurfactantare larger values; for example, the di-sodium salt of spiculisporic acid: 98.8A2 (100) and the sodium salt of rham-nolipid B: 79.1 ;"2 (19), respectively,at the air-solution interface from sur-face tension vs. concentration plots incomparison with 60 ;"2 for poly-oxyethylene [10.5 units] non yl-phenylether; 82 A2 for polyoxyethe-lene [20 units] nonylphenylelher(101), 68 ;"2 for aescin (102), asdetermined from the -r-c relationbelow the CMC. However, biosurfac-tants have smaller CMC values (I)because of the ease of molecular ori-entations.

Optical activity and secondarystructure formation also are uniquecharacteristics. Both of these charac-teristics may be attributed as physio-logical activities. The effects of the

Rhamnalipid B (RB)

NoO)S-O-CH2 -ICH2),o-CH3

~Sodium cccecyrsurtcte (SOS)

Figure 1. COmpllrison of the bulkln ... e ofrhemnollpld B.I the blolur1actlnt withsodium dodecytsulfale (SDS) In the CPKmode'

INFORM. Vol. 4. no.10 (October 1993)

1159

1160

I OLEOCHEMICALS

chirality of surfactants on surface-active properties have not always beenclear (103). Recently we correlatedthe emulsifying action of partiallyaeylated hyaluronates (av, mol wts 2-200 x 104, respectively) for soybeanoil to the u-hetix formation of thepolymer chains, as indicated by thedegree of polymerization of acyl-hyaluronates and the enhancement ofthe complex formation with toluidineblue (104). It was determined that thelarger the molecular weights of acyl-hyaluronales, the greater the emulsify-ing actions are, different from the factthat smaller polymers generally showlarger emulsifying actions. It was con-cluded that the ordered and controlledpolymer chains produced by the helixformation improved their emulsifyingaction. Our group (105,106) also hasbeen working on the dispersion-floc-culation systems for the developmentof pH-responsive display devices. Forexample, the reversible flocculation ofpH-responsive anionic microgels (200nm. methylmcthacrylate/acrylic acidcopolymer) with poly-L-Iysine (molwt 10,000) was electrically controlledby a pH modulator. This researchapplied the working mechanism thatpolylysine molecules make the sys-tems involved by mediating thecrosslinking among anionic microgelsdue 10 the electrostatic attraction inacidic conditions. Conversely, the sys-tems become transparent in alkalineconditions owing to the desorption ofpolylysine, rapidly forming their ownhelix chains apart from the microgelsurfaces. The response (redispersion)time was less than a millisecond.

It is easy to understand the notablesurface activities from the small CMCvalues, y-CMC, and interfacial tensionlowering of rhamnolipids (18,19), tre-halose lipids (30). corynomycolic acid(70) and H-13A gJyceroglycolipids(91), previously described.

The multiple hydrophilic andhydrophobic moieties of bicsurfactarnmolecules are advantageous for thedesired functions. For example. rham-nolipid A and B have both rhamnoseand carboxylic moieties as thehydrophilic portions and branchedalkyl chains as hydrophobic portions.

Therefore, rhamnolipid A and Bcould be dissolved in water in a wide

range of pHs greater than pH 4.0. Avariety of molecular aggregates wereproduced including micelle, lipidicparticle, lamella to vesicle dependingon the environmental pH values(95,96). Alkylamine salts of spi-culispcric acid also showed a similarbehavior (106). They resulted in aque-ous gel (about 10 cp in 1.0% solution)on the basis of liquid crystal forma-tion. Therefore, they may be used asdispersants for carbon black, phthalo-cyanine, Fe304 and y-Fe203 etc. oremulsifiers for hydrocarbons, etc.,with the stabilizing actions due to gelformation. On the other hand, succi-noyl trehalose tetraester (37) was agood dispersant due to its multifunc-ricnallty. Meanwhile, the convertibili-ty of the functions of rhamnolipid Bwas examined by esterifying the freecarboxylic acid moiety to the methylester eRB-Me). The surface activitiesof the newly obtained nonionicRB-Me were changed considerably(107). The CMC and y-CMC ofRB-Me increased, while the interfa-cial tension was reduced. Further-more, the hemolytic action of RB-Mefor rabbit erythrocytes was reducedfrom the level of RB-Na to that ofsodium dodecylsulfate (50S).

Biosurfactants usually have goodbiodegradabilities. In fact, theBOOlThOO of sophorolipids was75% after an incubation period of 14days (108). Soaps showed 60-70%depending on the test conditions(108). Disodium salt of spiculispcricacid (109) and the derivative of 2-(2-carboxyethyl)-3-decylmaleic acid(spiculisporic acid anhydride) (110)showed levels of biodegradabilitysimilar to soaps.

The biosurf'actams concerned inour research discussed here are safesurfacterns. Several safety tests (LDso,LC50. irritations for skin and rabbiteyes. erc.) for the disodium salt of spi-culisporic acid were carried out, andLD50 was 2,168 mg/kg for mouse.Biological activities of some surfac-rants were described previously.

Strategy for applicationThe first approach aimed at the uti-lization of biosurfactants is theirapplication for fine chemicals. Thisapproach is based on the discovery of

1.0C,2-55

2130mol%

r=39mol%~.Omol%

h~:;t'-g<;W;i'Thoutff ~ C12-55

~ O.4mM~. _ coree in soln.

oil'i;;60:c'i1'!i20~'80~2d'40~300;;'Time (min]

Constitution of ncoscmes30trol% 20 10 .. 0 •

Egg lecithinCbclesterol

C'2-55Dep

50 60 70 70 7020 20202020-30 2010 - - -- - - 10 - -

Rgure 2. Effect of N,N'-palmltoyl-L-cystlnecontent of llposome on calcein treneteracross the keral1n membrane

new functions of biosurfactants asdescribed above. Sophorolipids, spi-culisporic acid (ca. $23/kg), agaricicacid, surfactin (ca. $90/mg), andemulsan, etc., are now commerciallyavailable because of their reasonableprices and specific functions. Bioin-dustrial and enzymatic processes forbiosurfactant production seem to be aspractical as chemical synthesis pro-cesses.

Secondly, it is important in prepar-ing biomimetic surfactant's to under-stand the chemical structures andfunctions of biosurfactants and natu-rally occurring amphiphites. In addi-tion, it is also useful to modify bio-logical raw materials such as carbo-hydrates, amino acids, peptides, lipidsand so on. As examples of the former,synthetic corynomycolic acid ana-logues were prepared (70). Syntheticcorynomycolic acids have the advan-tage of containing single alkyl chaincompounds. depending on the alkylchainlengths of the long-chain fattyacid esters used as raw materials. Onthe other hand, microbial corynomy-colic acids are mixtures of differentalkyl chain components. However,synthetic corynomycolic acids areequimolar mixtures of syn and antidiastereomers compared to aU amicompounds in the microbial acids.Subsequently, N-palmitoyl-OL-

1161

CH,GH

H~HOH

~CONH0'0-"0

/

D-glucosorrine N'ocyl derivativemp 194-196·C (dec.I,(oID t20.1

Sodiumsetts [IV]Alkylaminesens ~COOH

I r Acid~"'Ydr~. ~. HOO'ir> mp 40-41'C IC2H,J,Nl:§X.0:@TNIC,"~2

~O [m] HOOC C~qCOOH C'O

Splculisporic acid l4.5 ·cialrboJ y' 4· pentodeconolidempI46'C,[a)o-12.6 H,COOC

t n H ~O

j R COOCH,~ Dimethyl ester

~COOH (010-11.3

~ (VI]

Open-ring acidmpt32°C,[alo-2.6

t n:I

Sodium scltsAlkylamine salts

Rhodamine type derivative[V]

Figure 3. Synthetic scheme of lunctlonel chemlc.l, from eplcullsporlc acid

homosertnetectcne. etc. was preparedas a pH-sensitive surfactant precursorin acidic environment that was con-vertible to a carboxylic surfactant inalkaline conditions for the constitu-tion of an intelligent liposome systemfor use as a chemical sensor or a DDS(Ill). N.N' -dldodecyt-Lcystine alsowas prepared by the condensationreaction of L-cystine and lauroylchloride. as a redox-sensitive surfac-tant precursor (112). Also, this com-pound was studied as a mediator forthe percutaneous absorption of bio-logically active substances to the skinfor cosmetics or transdermal thera-peutic systems (113). The modelexperiment was carried out using anapparatus connecting two chambersby a keratin membrane. When N.N·-dipalmitoyl-L-cystine was incorporat-ed into the Iiposomnl bilayer along

with lecithin and cholesterol, Iiposo-malty entrapped calcein, as a modeldrug, transferred well across the ker-atin membrane, which functioned asthe skin model (Fig. 2). The mecha-nism may be attributed to a largeaffinity for keratin as the main com-ponent of epidermis.

The third approach is to utilize bio-surfactants as synthetic intermediates.We have successfully prepared amphi-pathically hybridized chemicals. Asan example. functional amphipathicdyes were prepared from pyrene andspiculisporic acid (114) or rhamno-lipid B (115). We estimated the fluidi-ty and polarity in the microenviron-mems. such as micellar cores. lipidbilayers of vesicles, surfaces of poly-mers and biomembranes and organicsolvents. by use of fluorescent spec-troscopy. using pyrenacylesters of spi-

culisporic acid and rhamnolipid B asthe fluorescent probe. In particular,the solubility parameters of organicsolvents may be related to themicroenvironmental fluidity usingthese amphipathic fluorescent probes.Syntheses of functional chemicals viaspiculisporic acid anhydride from spi-culisporic acid were carried ouraccording to the scheme shown in Fig-ure 3 (116).

Future aspectIt is notable that biosurfactants exhibitexcellent and composite applicationson the basis of their specific function-al moieties, multifunctionalities. mildproperties and bulky structures. It is akey 10 progress in biosurfactants thatresearchers fmd new valuable applica-tions for contributions to the quality ofhuman life, environmental protection.

INFORM. Vol. 4. no. 10 (October 1993)

1162

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Kamala, 1. Jpn, Oil Chem. Soc.(Yukagaku) 36:791 (1987).

19. Itoh, S., and T. Suzuki, Agric.Bioi. Chem. 36:2233 (1972).

20. Nakahara, T., K. Hisatsuka and Y.Minoda, J. Ferment. Technol,5NI5 (1981).

21. Matsuyama, T., K. Kaneda, I.lshizuka, F. Toida and I. Yano, 1.Bacterial. 172:3015 (1981).

22. Syldatk C., S. Lang, U. Mu+ulovic. and F. Wagner, Z. Natur-forsch 40c:61 (1985).

23. Koch, A.K., J. Reiser, O. KappeJiand A. Fiechter, Biotechnology6(ll).-1335 (1988).

24. Parra, J.L., J. Pastor, F. Comelles,M.A. Manresa and M. Bosch,Tenside Surf 27:302 (1990).

25. MacElwee, e.G., H. Lee and J.T.Trevors, J. Ind. Microbial. 5:25(1990).

26. Bloch, H., I, Exp. Med. 91:197(1950).

27. Lederer. E., Chern. Phys. Lipids16.-91 (1976).

28. Lederer, E., A. Adam, T. Cicr-barn, 1.-F. Petit and J. Wietzerbin,Mol. Cell Biochem. 7:30 (1975).

29. Suzuki, T., K. Tanaka, 1. Matsub-ara and S. Kinoshita. Agric. Bioi.Chern. 33:1619 (1969).

30. Kretschmer, A., H. Bock and F.Wagner, Appl. Environ. Microbi-01.44:864 (1982).

31. Rapp, P., H. Bock, V. Wray and F.Wagner, J. Gen. Microbiol. l/5:491 (1979).

32. Wagner. F.. J.-S. Kim. S. Lang,Z.-Y. u,G. Maravede, U. Matur-ovic and E. Ristau, The 3rd Euro-pean Congress on Biotechnology,Vol. 1, 1984, p. 3.

33. Batrakov, S.O .• B. V. Rozynov.T.V. Kollonelli and L.D. Bergel-son, Chern. Phys. Lipids 29:241(1981).

34. Uchida, Y, R. Tsuchiya, M.Chino, J. Hirano and T. Tabuchi,Agric. Biot. Chern. 53:757(1989).

35. Uchida, Y., Thesis, Division ofApplieed Biochemistry. Universi-ty of Tsukuba (Japan), 1988.

36. Uchida. Y., S. Misawa, T. Naka-hara and T. Tabuchi, Agric. Bioi.Chern. 53:765 (1989).

(comtnued on page 1164)

For Information circle 1255

biodegradable substances, resourcesand energy. as well as for the surfac-tant industry. The recent developmentof both bioindustrial and chemicalsynthetic processes may support acontinuing supply of biosurfactantcandidates. On the other hand. high-priced and tailor-made surfactants,and intelligent surfactant systems, aredesirable to learn the important rolesof amphipathic or surfactant con-stituents in the biologicaJ systems.

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71. Tabuchi, T., I. Nakamura and T.Kobayashi, 1. Ferment, Technol.55."37 (1977).

72. Tabuchi, T., L Nakamura and E.Higashi, Ibid. 55:43 (1977).

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74. Matsuyama, T., K. Kaneda, I.Ishizuka, T. Toida and I. Yano, 1.Bacteriol. 172:3015 (1990).

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95. Ishigami, Y., Y. Gama and S.Yamazaki, Ibid. 36:490 (1987).

INFORM. Vol. 4. rc.to (October 1993)

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96. Ishigami, Y., Y. Gama, H. Naga-hera, M. Yamaguchi, H. Naka-hara and T. Kenara, Chern. Lett. p.763 (1987).

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107.Ishigami, Y, Y Gama, F. Ishiiand Y.K. Chic, Langmuir 9(6), inpress.

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llO.Choi Y.K., CoH. Lee, Y. Takaza-wa, Y. Gama and Y. lshigami, l.lpn. Oil Chern. Soc. (Yukagaku)42 ..95 (1993).

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114.1shigami, Y., Y. Gama and S.Matsuzaki, l. lpn. Soc. ColourMaterial (Sikizai) 64:431(1991).

115.Ishigami, Y., Y. Gama and S.Y.Matsuzaki, Ibid. 62:594 (1989).

116.Ishigami, Y., Y. Gama, S.Yamazali and S. Suxuku, in Pro-ceedings, World Conference onBiotechnology for the Fats andOils Industry, edited by T.H.Applewhite, AOCS, Champaign,Illinois, 1987, p. 339. •

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