lipase e monoacilglicerois

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Lipase syntheses of monoacylglycero ls: U. T. BornscheuerTable 1 Applica tion of monoacylglycerol

Application References

Cosmetics, toothpastes, etc.Emulsifier, mainly in food

technologyMedicine and therapyHair care additive

Baumann et al., 3s Sonntag3gKott ing and EibP5

Li and Ward, a Akoh et aLg8Weiss4

The best results were obtained at pH 7.5 with 23% M AG(mainly monoricinolein) and 66% ricinoleic acid after 3 hreaction time. Precipita tion of the acid by the addition ofsalt raised the MAG content up to 65%. To investigate thestructural properties of the active site of lipase B fromPseudomonas fragi 22.39B, glycerides were hydrolyzed byTsuzuki et al. in a homogeneous solvent system (buffer-tetrahydrofuran). The lipases were made soluble in organicsolvents and the effect of the chain length and number ofunsaturated bonds were investigated.61

alternative process for the production of fatty acids undermild conditions. Severa l methods have been described inthe literature, including reactions in the presence4& orabsence 52*53of organic solvents, and examples for contin-uous hydrolysis employing different reactor systems (e.g.,hollow-fiber bioreactors). 54-57The latter examples were re-viewed by Mukherjee.37To obtain MAG by enzymatic hydrolysis, it is necessaryto control the reaction so that complete hydro lysis isavoided. The easiest way is the application of 1,3-sn-specific lipases yielding 2-MAG [Scheme I, Eq. (l)]. Themain problems in this type of reaction are the low overal lyields of MAG, because 2 mol free fatty acid mol- MAGis produced and acyl migration [from the 2 to the l(3)position of glycerol] has to be suppressed. Partial hydrolysiswas achieved for some seed oils, such as mustard, crambe,and honesty.58,59 Flenker and SpenefiO hydrolyzed castoroil by using a 1,3-specific lipase from Rhizopus arrhizus.

A more efficient preparation of 2-MAG in a biphasicsystem was presented by Mazur et a1.62 The system con-sisted of hexane, aliphatic alcohol (e.g., 2-butanol), and anaqueous buffer containing 1,3-specific lipase (lipozyme). Itwas found that higher selectiv ity was induced by the ester-ification of the released free fatty ac ids with the aliphaticalcohol (namely primary alcohols) in a second step. Theyield was 70%, and the method was applicable for trig lyc-erides from C,:, to C,4:0.62 Another poss ibility for the in-duction of selectivity was achieved by the hydrolysis of fatsin oil-rich microemulsions in the presence of anionic sur-factants. Holmberg and &terberg63 were able to obtain2-MAG in 80% yield under optimum conditions. However,slow acyl migration was reported and higher yields wereimpossible because of complete hydrolysis to fatty acid andglycerol.The problem of acyl migration and the stereo- and re-gioselectivity of lipases in reactions of glycerol derivativeswere investigated by severa l groups646R and are beyond thescope of this article.

OR

hRO OR

Fat/Od

ORl.3-sn-speclfic l1pase.n

(1)2 R-COOH

HO OH

Esterification of fatty acids or esters with glycerol

2-MAC

OH(R)

Fatty sadH& OR

I(2)-MAC

R~oxR

The discovery of the high stability of lipases in organicsolvents offered the possib ility of a reverse reaction of hy-drolysis, esterification using free fatty acids (FFA), ortransesterification using esters [Scheme 1, (Eqs . 2 and 3)].For that purpose conditions are needed, in which the enzymewil l catalyze the synthesis reaction rather than the hydroly-sis. Most important, low water content and low water ac-tivity are necessary. 69-71To achieve a high-yield formationof MAG and the suppression of unde$ired hydrolysis , sev-eral methods have been described, as shown in Table 2.Water generated during the reaction was removed by theaddition of molecular s ieves *, or reactions under reducedpressure.74 Examples for the synthesis of MAG by esteri-fication are listed in Table 3 and are discussed in moredetail.Ester

OH(R)IpW

h(3)- K-OH

HO OR(H)I(2)-MAC

Influence of the fatty-acid chain lengthOR

ARO OR

OH OH(R)IlpW- A (4)

Fat/OdH& dR8 HII(2)-MAC

Scheme 1 Schematic drawing of lipase-catalyzed methods forthe synthesis of monoacylglycerol (MAGI using selective hydro-lysis with 1,3-sn-specific lipases [eq. (I)], esterification of fattyacids [eq. (2)] or esters [eq. (3)] with glycerol and the glycero-lysis of fats oils [eq. (4)]. By-products such as diacylglycerol,triacylglycerol, and free fatty acid are not shown, for clarity

In 1977, Tsujisaka et al. 7s demonstrated the versa tility oflipases for the esterification reaction. With lipases fromAspergillus niger or Rhizopus delernar, esterification ofoleic acid occurred almost exclusiv ely at the sn-1 or sn-3positions of glycerol . On the other hand, lipases from Geo-trichum candidum and Penicillium cyclopium esterifiedonly long-chain fatty acids, and esterificat ion occurred ran-domly.75 The influence of the fatty-acid chain length wasalso studied by other groups. 7G78 Jansisenet al. 76 found thata higher monoester formation was possible with short-chainEnzyme Microb. Technol., 1995, vol. 17, July 579


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ReviewTable 2 Methods to achieve a selective formation of MAG inesterification reactions

Method Principle References

Addit ion of molec-ular sieves

Reduced pressureAdsorption of

glycerol on sup-port material

Adsorption ofMAG on in- l inecolumnAddit ion of phe-nylboronic acid

Reaction in solidphase

Water removal

Water removalShif t of equi l ib-

rium, enhancedselectivity

Shif t of equi l ib-rium, enhancedselectivityEnhanced solubi l -ity of glycerol,protective agent

Shif t of equi l ib-rium

Yamaguchi andMase,72 Gan-ceF3

Mil ler et a/.74Berger and

Schneider87,89Van der Padt etalkslSteffen et a/.92Weiss4

fatty acids in polar solvents, whereas a higher d i- and tri-ester synthesis took place with longer-chain fatty acids innonpolar o rganic solvents. The ester molar fractions at equi-librium were found to depend on the fatty chain length inthe absence of a solvent. A computer program (Two-phaseReaction Equilibrium Prediction) was used to predict equi-librium amounts of fatty acids and esters formed.A high-yield synthesis of MAG was achieved by theaddition of molecular sieves.72 Under optimal conditionsthe conversion of oleic acid was 97.3% and 74 wt% MAGwere formed by a lipase from Penicillium camembertii.Long-chain unsaturated and medium-chain saturated fattyacids were efficient acyl donors. The conversion of diacyl-glycerol (DAG) to MAG was also observed by the additionof molecular sieves.Monooleylglycerol as a model MAGMonoo leylglycerol has been used by many research groupsas a model MAG. Surprisingly, the achieved conversionsand selec tivit ies differed significantly (Table 3) dependingon the source of lipase, the reaction system, and other pa-rameters .Schuch and Mukherjee 79 found that with lipase G (Peni-cillium sp.), higher reaction rates and a higher formation ofmonooleylglycerol were achieved compared to reactionscatalyzed by lipozyme, in which dioleyl- and trioleylglyc-erol were the most abundant products. They also investigatedesterification with [l-4C]-labeled oleic acid with mono-and dioley lglyce rol and the hydro lysis of [ 1 14C]-labeledtrioleylglycerol.The formation of MAG in tertioamylic alcohol, trichlo-rotrifluoroethane, and supercritica l carbon dioxide bymycelium lipase from R. arrhizus was reported by Gan-cet.73 Whereas in tertioam ylic alcohol no formation ofDAG was observed (17.9% MAG), in trichlorotrifluoro-ethane almost simila r amounts of MAG (37.10/o), DAG(30.5%), and oleic acid (32.4%) were detected. The highestMAG content (44.1%) was achieved in supercritica l carbondioxide at 77 bar and 32C.73 The reactions in tertioamy lic

alcohol were also performed in a continuous two-fixed-bedsegment with dehydration of the reactional medium be-tween each pass. At a residence time of 1 h between eachpass, 44.7% MAG was formed almost exclusively and ahigh stability of the lipase was reported.The influence of organic solvents on the synthesis ofoleylglycerols was recently studied by Pastor et a1.80 andOtero et al. The direct esterification of oleic acid withglycerol in n-heptane gave only a low MAG content (max.7.2%) even at high temperatures (max. 70C). The trans-esterificat ion of ethyl oleate with glycero l in different sol-vents led to a higher formation of MAG, especially withoutthe addition of water. Here, at optimized conditions up to68%, MAG were formed after 2 h by lipase from Candidaantarctica in acetone. Furthermore, at short reaction times,it was found that the reaction was faster and more selectiveto MAG production when a polar solvent (e.g., acetonitri le)was used.Reactions in reverse micellesThe examples explained above were performed in solvent-free systems or in the presence of organic solvents . Asalternative reaction systems, reverse micelles were alsoused for the lipase-catalyzed formation of MAG as reactionmedia.82-86 Besides the advantages of a high interfacialarea, easy preparation, and good enzyme-substrate contact,the encapsulation of glycerol into the reverse micelles over-comes the problem of the low solub ility of glycerol in or-ganic solvents.Hayes and Gularis4 employed a 1,3-specific lipase fromR. delemar, but mainly l-MA G was formed. This observa-tion was related to an acyl migration of a fatty-acid residuefrom the 2- to the l(3)-posit ion of glycerol. The conversionreached S&60%, and the reaction rate decreased in theorder: oleic acid > caprylic acid > myristic acid = lauricacid > stearic acid = palmitic acid. A higher conversion(80-90%) was reported by Fletcher et a1.,82 but the lipaseemployed was not 1,3-positional-specific and both l- and2-MAG were formed. This was also reported by Bom-scheuer et al. 85 for the reaction between glycerol and lauricacid using purified lipase from Pseudomonas cepacia inAOT (Dioctylsodiumsulfoccinate) microemulsions.Whereas lipase from Chromobacterium viscosum was foundvery stable in AOT reverse micelles,** R. delemar lipaseshowed only poor stability in the same system.84Systems based on selective adsorptionThe high-yield synthesis of regioisomerically pure 1(3)-MAG in organic so lvents on a multigram scale was reportedby Berger and Schneider. 87-90The problem of the low sol-ubility of the hydrophilic glycerol in nonpolar organic sol-vents was overcome easily by prior adsorption of glycerolonto a solid support (e.g., silica gel, charcoal, ce lite) cre-ating a dry powder. These glycerol preparations were sus-pended in an organic solvent (e.g., t-butylmethylether, di-ethylether-hexane) and an acyl donor together with a 1,3-select ive lipase (e.g., R. delemar, Rh izomucor miehei,Chromobacterium viscosum) were added, thus mimickingan artif icial liquid -liquid interphase. After the reaction was

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Lipase syntheses of monoacylglycerols: U. T. BornscheuerTable 3 Synthesis of MAG by lipase-catalyzed esterification reactions

Lipase Substrate Main product0 ReferencesMucor miehei OAAspergillus niger, Rhizopus

delemar, Geotrichum candi-dum, Penici l l ium cyclopiumPenici l l ium camembert i i

Penici l l ium sp., LipozymeRhizopus arrhizusCandida antarcticaRhisopus delemarLipozymeLipozymeGeotrichum candidum,

Pseudomon as sp.. Mucormiehei

Penicillium cyclopium, Rhizo-pus sp.

Chromobacterium viscosumHumicola lanuginosaAspergillus nigerRhizopus delemar, Mucor mie-

hei, Chromobacterium visco-sumCandida rugosa

OA

OAOAOAOA, ethyloleateOA and othersOA, SAeg., (S)-17-hydroxystearic acidEPA, DHA

Solid FAA (e.g., C,,)G-G,, cl*:,G-Goc4-c,,, h3:,. %3:*e.g., C&,-acids

(sat. and unsat.)Caprylic acid

l-MAG (max. 32%),80% conv.

l,(3)-MAG or random

MAG (max. 74% wt)Low to high MAGMAG (17.9-44.1%)MAG (7.268%)l-MAG, 50-60% conv.MAG, 30-70% conv.MAG (max. 84%)6.4-65% MAG

MAG (max 96%)High MAGMA GMA Gl,(3)-MAG, >70%yieldMAG >90%

Yamane et al . ,93 Hoq et al .94Tsujisaka et al.75

Yamaguchi and Mase7*Schuch ahd Mukherjee79Gancet73Pastor et al.*OHayes and Gularis4Singh et al .86Steffen et al.92Li and Ward,40 Akoh et al.,g6

Osada et aLg7Weiss?Janssen et al.76IbrahimTahoun et a/.sBerger and Schneidera7,sg

Van der Padt et al .9q

OA, oleic acid; SA, stearic acid; FA, fatty acid; EPA , eicosapentaenoic acid; DHA, docosahexaenoic acid; $at., saturated; unsat.,unsaturated; max., maximu m; conv,. conversion

completed, both the enzyme and the solid support wereremoved simultaneously by simple filtration while the es-terification products remained in solution. Under optimizedconditions, the MAG content achieved 70%, but higheryields were found to be impossib le, because at high con-centrations, l(3)-MAG served as a better substrate thanglycero l. x7 For a higher yield, the authors developed a con-tinuous separation system based on the compartmental sep-aration of the two steps of the process: synthesis and isola-tion . Unreacted substrates and unwanted by-products [e .g . ,2-MAG, DAG, triacylglycerol (TAG)] were completely re-cycled and l(3)-MAG was frozen at lower temperatures(Scheme 2). Here, also , nonspecific lipases were used suc-cessfully.87The successof the method of Berger and Schneiders wasRecychng

WOH i OH Ih

+ I eq. acyl donor bpt3.X_h

+ GlyceridesHO OH HO OAcyl

Glycerol(adsorbed1

1 (3).m-monoacylglycerol

Scheme 2 Synthesis of regioisomerically pure l(3)-mono-acylglycerol (MAGI using adsorbed glycerol; isolation and recy-cling of by-products (according to Berger and Schneider3?

demonstrated by using numerous acyl donors, such as freefatty ac ids (from C, to C,, in saturated and unsaturatedconditions), fatty-acid methyl esters, vinyl esters, synthetictriacylglycerols, and natural fats and oils. Various solidmonoacylglycerols were produced in high yields and iso-meric purity without the need for further purification steps.An alternative process was based on an immobilized en-zyme membrane reactor equipped with an in-line adsorptioncolumn.91 In this reactor, an internal oil circu it (containingthe fatty acid and produced glycerides)i and an external glyc -erol-water circu it are kept separated using a hollow-fibermembrane module. The lipase is adsorbed at the inner fiberside and an in-line column is placed in the oil phase circu itof the reactor. Van der Padt et ~1.~ $ound that monoacyl-glycerols (e.g., monocaprinate) adsorbed preferent ially onthe in-line column, thus removing MAG from the reactionmixture, shifting the equilibrium and repressing the furtheresterification to DAG and TAG. A high purification factor(>90%) was achieved by mild (off-line) elution from com-petitively adsorbed compounds such as DAG and free fattyacids. The authors implied that the membrane reactor can beused in a continuous process using a sequence of columns.They estimated a production of 60 mol (15 kg) monoesterg ~ enzyme. The half-life time of the lipase from Candidarugosa was given as 50 days.Other systemsThe additive, phenylboronic acid (PI&A), was described asa solub ilizing and protecting agent in the esterification of

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Reviewglycerol with uncommon fatty ac ids in the presence of or-ganic solvents.92 The highest yield of MAG (84%) wasobtained using (S)-17-hydroxystearic acid as the substrateand lipozyme as the biocata lyst in n-hexane. Steffen et a1.92found that MAG were produced exclus ively at a high ratioof free fatty acid (PPA):glyce rol:PBA (e.g., 1:5:5); 12-hydroxy-stearic acid, 9(10)-acetonyl-stearic acid, and9( 10)dimethylmalony lstearic acid served as further sub-strates .Another method was based on the enzymatic esterifi ca-tion of glycerol with solid fatty acids and their methyl estersemploying lipases from Penicillium cyclopium or RhizopusSP.~~The reaction mixture consisted of the fatty acid dis-persed as fine, solid particles in the lipase-containing glyc-erol phase. The system was applied for the synthesis ofmonopentadecanoylglycerol, a hair care additive. Underoptimized conditions almost quantitative conversion of thePFA to the corresponding MAG (96%) was achieved. Theselec tivity of the lipases differed greatly, which was dem-onstrated for the reaction of palmitic acid with glycerol.With lipase from P. cyclopium, up to 85% MAG was syn-thesized, whereas under identica l reaction conditions, lipasefrom Rhizopus sp. yielded only 5-8% MAG, and DAG wasthe main product. Disadvantages of the process describedby Weiss41 were the thermal inactivation of the lipases ,which took place in the downstream process, and the largeexcess of glycerol.The continuous production of glycer ides from liquidfatty acids and glycerol by lipase from Mucor miehei in amicroporous, hydrophobic membrane reactor was reportedby Yamane et a1.93 and Hoq et a1.94 Thirty-two percentMAG (mainly l-MAG) was produced at 80% conversion.The half-life of the enzyme was extrapolated to 54 days.94The application of membrane bioreactors for the esterifi ca-tion of fatty acids with glycerol and the hydrolysis of oilshas been comprehensively reviewed by Kloosterman eta1.95Of special interest for pharmaceutic application are n-3polyunsaturated fatty acids such as eicosapentaenoic acid(EPA ) and docosahexaenoic acid (DHA) (Table I). EPAand DHA are only completely adsorbed as glycerides suchas monoacylglycerols.40 Their synthesis was performed byesterificat ion with lipases from Geotrichum candidum,96-97Pseudomonas sp . , or M. miehei.40 The reaction systemswere optimized with respect to water and glycerol content,organic solvent, temperature, and other conditions. Thecontent of MAG varied widely, from 34.5% MAG (mainlyEPA and DHA-MAG40) to 65% E PA yield96 and 6.4%(DHA-MAG) to 8.65% (EPA-MAG).97

Synthesis of MAG using protected glycerolTo overcome the problem of subsequent acylation, whichmay occur in esterifications with pure glycerol, several re-search groups employed protected glycerols. This also of-fered an advantage for the synthesis of posit ion-specificand/or enantiomerically pure, or at least enriched, MAG.

Usually, a fatty acid was enzymatically esterified with aderivatized glycerol moiety that had two of the three hy-droxyl positions blocked (Scheme 3). Once esterified, theblocking group then was removed in a subsequent step,resulting in the formation of regioisomer ically pure MAG.As discussed in the previous section, one problem wasthe removal of water generated during esterification. Thiswas achieved by using open test tubes,98 reactions underreduced pressure,99 or by the addition of molecula rsieves . loo By controlling substrate ratio, type of acy l donor,cleavage catalyst, and concentration, it was possible to syn-thesize MAG in a purity of up to 100%.Severa l reports have dealt with the esterifica tion of oleicacid with 1,2-0-rat-isopropylidene-sn-glycero l (1,2-0-IPG; 2,2-dimethyl-1,3-dioxolane-4-methanol) catalyzed byimmobilized lipase from M. miehei (lipozyme).98-*00 Al-though the authors investigated the same system, theachieved conversions, yie lds, and optimum temperaturesdiffered greatly. The reason may be the use of differentamounts of lipozyme, substrate ratios, and reaction times.Pecn ik and Knez99 reported a strong influence of pressureand temperature. Optimum conditions were found at 0.057bar and 55C, resulting in 80% conversion of oleic acidafter 350 min reaction time. They also determined thermo-dynamic properties and reported that the energy of activa-tion was found to be temperature-independent in the interva lfrom 10-70C.99 Omar et al. loo reported the highest con-versions (>90%) at an oleic acid-IPG ratio >3. loo Thehalf-life of lipozyme was calculated to 20 days, and theproductivity was 869 g product g - lipase. The specificityof lipozyme toward other fatty acids with cha in lengthsfrom C,, to C,, and C1sL2 o C,,:, were found to be simi larto those found for oleic acid (C,,:,). HeB et al.lo l synthe-sized l-monostearoylglyceride from either stearic acid orethyl stearate with 1,2-0-IPG and lipase from Pseudomonascepacia followed by hydro lysis of the acetonides with bor icacid. The highest reaction rates were found in n-heptane orn-dodecane; the maximum conversion was 84%. Increasingthe alkyl chain length of stearate led to a decrease in thereaction rates. The remaining activ ity of the lipase was veryhigh, up to 64% after 24 days incubation time was deter-mined. The authors also observed an enantiomeric excess

;x;p Ok

0selective hydrolysis

_ J * Ho)+ A lR HO

1.2-O-protected glycerol fatty acid ester 1.2-O-protected-glycerol-fatty ester 3 .MAG

Scheme 3 Synthesis of monoacylglycerol (MAG) from protected glycerols and esters followed by cleavage of the protective group

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Lipase syntheses of monoacylglycero ls: U. T. Bornscheuer(%ee) of 11 for 1-monostearoylglycerols synthesized fromethyl stearate and 1,2-0-IPG.Another approach in the synthesis of MAG with IPG wasbased on the transesterifica tion employing either meth-yloleate in n-hexaneg8 or the application of enol ester. 2*03In the former case, the conversion ranged from 46.9-67.8%depending on the lipase type and amount of solvent. Afteracid catalyzed cleavage of the protecting group, between27.1 and 50.3% MAG was determined. Another attemptwas based on the application of enol esters, which offer theadvantage of a shift of the reaction equilibrium due to irre-versib le tautomerization of the by-product v iny l alcohol(yielding acetaldehyde) or isopropenyl alcohol (yielding ac-etone). Bomscheuer and Yamane achieved 100% con-version of long-chain fatty-acid vinyl esters (vinyl decano-ate, laurate, palmitate, and stearate) to the correspondingacetonides in short reaction times (up to 24 h) in a solvent-free sys tem or in the presence of n-pentane. Wang et al.produced glycerol monovalerate and glycerol monoacetateusing the same process. Moreover, these monoacylglycer-01s have been enantiomerically enriched, and up to 67%eewas achieved. In this reaction, which was also catalyzed bylipase from P. cepacia, the unreacted IPG was almost enan-tiomerically pure (92%ee). lo3An important step in the reaction employing blockedglycerols is the cleavage of the protecting group. V ariousmethods have been investigated, and ranged from mild hy -drolysis employing boric acid in 2-methoxyethanol oriodine in methanollo to mean and strong acidic conditionsusing concentrated HCl o r trifluoroacetic acid.98,99.02 Un-fortunately, strong acidic conditions resulted in the interes-terification between two MAG molecules, leading to theformation of DAG and glycerol as by-products and acylmigration.98.102The influence of the protecting group on the reaction rateand optical purity was investigated for the enzymatic hy-drolysis of different 1,2-ketals of glycerol.106 The highestselectivity was found for the hydrolysis of 2,2-dimethyl-1,3-dioxolane-4-methanol butanoate yielding glycerolmonobutanoate and catalyzed by proteinase from Aspergil -lus oryzae. The umeacted ester had an enantiomeric excessof 84%, and the E value was calculated at 9.0. Other pro-tecting groups were fluorenone, Spiro, and diphenyl com-pounds but only in the latter case was a sufficient reactionrate and enantiomeric excess (68%ee, E = 8.1) found.Surprisingly, hydrolysis of longer-chain fatty-acid deriva-tives gave no reaction; also, acetates or octanoate gave onlypoor results. lo6GlycerolysisAs outlined previously, the glycerolysis of fats and oilsemploying alkaline catalyst represents the process currentlyused in industry for the large-scale synthesis of MAG.Enzymatic glyce rolysis offers the advantage of a highspace-time yield because 3 mol monoacylglycerols mall triacylglycerol could be formed [Scheme 1, eq. (4)],whereas in the hydrolysis of triacylglycerols with 1,3-specific lipases 2 mol fatty acid is synthesized per mole ofMAG.Initiated by the work of Yamane et al., lo7 lipase-

catalyzed glycerolysis received considerable attention, andseveral papers dealt with the glycerolysis reaction of modelfats and a wide variety of natural fats and oils. The reactionswere performed in reverse micelles,108~09 in the pres-enceO. l1 or absence * of organic solvents, and in a sol-vent-free-solid-state system. 13- I6 Problems employingthe hydrophilic substrate glycerol in organic solvents andanalyt ical problems for the quantitative determination ofpartial glycer ides were described by Ferreira -Dias and Fon-seca. Yamane et u1.07 studied the glycerolysis reaction ofcorn oil by Pseudomonusfluorescens lipase in a batch sys-tem without the addition of surfactants or emuls ifiers. At40C and 3.7% water content in the glycerol phase, 20.4%MAG content was determined. It was also reported thatlipase was inhibited and inactivated by fat peroxide in thecourse of batch and continuous glycerolyses of saffloweroi1.18 Other authors3m6,R improved their system bycarrying out the reactions first in a liquid-liquid emulsionstate, then cooled until the glyceride-glycerol mixture be-came solid. The strong inc rease in the formation of MAGafter reaching solid-s tate was related to the crysta llizat ion ofMAG from the reaction mixture. This led to a shift in thereaction equilibrium. Using this method, a high (70-99%)formation of MAG from various natural fats and oils such asbeef tallow, palm oil, palm stearin, palm olein, coconut oil,rapeseed oil, and hydrogenated tallow and lard was re-ported. The most important parameter in this process wasthe reaction temperature, and much care was taken for thedetermination of optimal temperature programming. Theauthors created the term critical temperature (Tc) for thesharp transition temperature between the high and lowmonoglyceride states. Further optimization of the reactionand an increase of MAG content were achieved by control-ling the water content, the ratio of triacylglycerol to glyc-erol, and the amount and type of lipase. l4 Activities andstabilities of lipases from various sources were recentlystudied using triolein as a model triacylglycerol. It wasfound that bacterial lipases were very stable, whereas yeas tand mold lipases were quickly inactivated.s A further in-crease of the operational stabil ity was achieved by immo-bilization of lipase on Celite. With lipase from Chromobuc-terium v iscosum 99% MAG content was achieved after in-cubation for 120 h at 8C (first 8 h at 25C).A correlation between fat composition and MAG yieldby means of chain length and saturation pro file of the fattyacids was found in the solid-state glycero lysis of palm oil-based fats and blends with rapeseed oil. The detailed com-positional analysis of the reaction products strongly indi-cated the preferential crysta llizat ion of MAG, 19, *O espe-cia lly for the accumulation of saturated fatty ac ids such asC,,:, in the MAG fraction. Optima l cond itions were foundat a saturated fatty-acid content of 20180% of the originalfat, whereas at a content >80%, the formation of DAG wasfavored. McNeil1 and Berger19 claimed that the yield ofMAG from a reaction carried out below the Tc can bepredicted from the fatty-acid composiit ion of the substrateusing simple linear regression analysi,s.Stevenson et al. l*l reported the glycerolysis of tallow insolid-state with the immobilized lipase lipozyme in a batchand a continuous system. The yield of MAG was 35% in theEnzyme Microb. Technol., 1995, vol. 17, July 583


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Review(solid-sta te) batch system. For continuous production, theliquid reaction m ixture was pumped through a column fi lledwith lipozyme, resulting in a decrease in the MAG yield,because crysta llizat ion of MAG had to be avoided. An in-crease in the overall MAG yield up to 69% was achieved byextraction of MAG into n-hexane, filtration of the enzyme,and recyc ling of the nonmonoglyceride portion with freshtallow in another reaction.Other systems for continuous glycerolysis were per-formed using a membrane b ioreactor22 or lipase immobi-lized on liposome in reverse micelles. lo8 In the microporoushydrophobic membrane bioreactor, edible oil was glycero -lyzed by lipase from P. fluorescens, resulting in the forma-tion of 5-25% MAG. The half-life of the enzyme was 3weeks at 40C.122 Chang et ~1.~~~employed a continuousstirred-vessel bioreactor with polysulfone membrane for theglycerolysis of olive o il in AOT/iso-octane reverse micellesusing immobilized lipase from C. viscosum. The lipase wasimmobilized on liposome (small unilamellar vesicle) as amatrix. The polysulfone membrane was 12.5 km thick , andthe pore diameter was a maximum of 25 nm. After a l-daydelay, a steady state was achieved, result ing in 80% con-version of olive o il during 6 days of operation. The highestproduc tivity of 1-monoolein (90 pmol h- ml - outlet)was obtained at 2.0% (v/v) of in itial substrate concentra-tion. Optimum flow rate of olive oil was 2.5 ml hh, andthe water content was below 8% (w/v). The operationalstab ility of the liposome-lipase was extrapolated to be 45days, whereas the half-life of the free enzyme in reversemice lles was only about 4 days. In the latter example, theactivity of the immobilized enzyme was only slightly lowercompared to the free biocatalys t.08 A two-fixed-bed seg-ment for the continuous glycerolysis of beef tallow wasdeveloped by Gancet .73 At a residence time of 75 min, ayield of 38.8 g MAG 100 g- beef tallow was reported.The reactor system was run for severa l months with a rathergood operational stabi lity of the myce lial lipase from R.arrhizus.Another h igh-yield formation of MAG was based on thepresence of a secondary or tertiary alcohol in the reactionmixture acting as solvent for all components including glyc-erol. A wide variety of natural fats and oils were glyc-erolyzed in the presence of t-butanol. After 3 h reaction at7OC, 82-90% MAG was synthesized by the immobilizedthermostable lipase from Candidu antarctica . The systemwas also applied for esterif ication and transesterificationreactions using fatty acids and their derivatives from C, toC 22.

Likewise in reverse micelles, Holmberg et al.loy inves-tigated the glycerolysis of palm oil using 1,3-specific li-pases. Previously, they showed that lipases were able tosynthesize MAG in a reverse-micelle system.63 They ex-pected that by partia lly replacing water with glycerol in thereverse-micelle system, selective glycerolysis would favorhydro lysis. In addition, MAG would be formed withoutconcomitant liberation of fatty acid. Although a maximumtheoretical yield of 3 mol MAG mall triglyceride wasexpected, the ratio of MAG to TAG was approximately 1.4.The authors suggested two reasons: competing hydro lysis(yielding 2 mol fatty acids 1 mol- TAG) and acyl migra-tion. To verify the former assumption, 3H-labeled glycerol584 Enzyme Microb. Technol., 1995, vol. 17, July

was used. Whereas hydrolysis gives only unlabeled MAG,glycerolysis should lead to the formation of 2 mol of labeledand 1 mol of unlabeled MAG mol - of trig lyceride . Therate of glyce rolysis was found to approximate that of hy-droly sis. Regarding the second assumption (acy l migra-tion), 2-MAG should give I-MAG, which is regarded as asubstrate for a subsequent hydro lysis reaction, also reducingthe final MAG yield. From four 1,3-specific lipases tested,those from Rhizopus sp. gave almost similar results,whereas lipase from porcine pancreas was less active. lo9ConclusionsThe examples shown in this review demonstrate the diver-sity of lipases in a wide variety of reaction systems that havebeen used for the synthesis of an apparently simple reaction,the production of monoacylglycerides . Although so far nosystems presented here have been commercialized on anindustrial scale, the chances for a lipase-catalyzed synthesisof emulsifiers such as MAG are increasing, for several rea-sons. First, according to Fu1ch,23 the progress in geneticsand in process technology enables the enzyme industry tooffer products with improved properties and often at re-duced costs. Second, some newly developed high-yie ldformations of MAG shown here may become more com-petitive in standard chemical processes. Lower energy con-sumption, mild conditions, higher space-time yields andselectivities, and better product quality should be men-tioned. In addition, the demands for green technologiesand consumer-friendly products are increasing , and bet-ter acceptance should be possible.On the other hand, a prediction of lipase-ca talyzed reac-tions with respect to substrate specif ity, time, and yield, forexample, i s still difficult. Tedious and skillful experimentsseem to be necessary for the development and optimizationof a new process or method. From an optimistic point ofview, these problems may be reduced in the near future asa result of new developments and further knowledge of themechanisms and actions of lipase under natural and unnat-ural conditions.AcknowledgmentThe author thanks Prof. T. Scheper for discussion and re-vision of the manuscript.References

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