Lipase-catalyzed syntheses

of monoacylglycerols
Uwe T. Bornscheuer
Institut fiir Technische Biochemie, Universitiit Stuttgart, Stuttgart, Germany

The application of lipases for the production of monoacylglycerols is reviewed. Glycerolysis or selective hydro-
lysis of fats and oils, esterification of fatty acids or esters with glycerol, and reactions employing protected
glycerols are presented. From the most recent literature, reaction systems ranging from organic solvents to
reverse micelles and solid-phase systems, for example, are covered, including examples for the continuous
production of partial glycerides.

Keywords: Esterification; glycerolysis; hydrolysis; lipase; monoacylglycerol; monoglyceride; organic solvent;

transesterification

Introduction people with different cardiovascular disorders.40 Monopen-

The application of lipases (triacylglycerol hydrolases
EC.3.1.1.3) for synthetic purposes is now well-
documented. In particular, the discovery made by Kli-
banov’-3 of their high stability and activity in organic sol-
vents has led to a strong increase in research activities to-
ward the synthesis of interesting precursors for organic
compounds and pharmaceuticals. These applications were
reviewed, and include aspects for industrial applica-
tions.416 In the area of fat research and technology, lipases
were employed successfully in the interesterification of fats
and oilsi7-23 for the production of high-value products
(e.g., cocoa butter), fatty acid ester synthesis,24 triglyceride
synthesis,25,26 synthesis of alkyl glycoside fatty acid es-
ters,27 the alcoholysis of fats and oils,28-33 and the produc-
tion of partial glycerides such as diacylglycerols3”36 or
monoacylglycerols. Lipase-catalyzed reactions for the mod-
ification of fats and other lipids were reviewed by Mukher-
jee.37
Monoacylglycerols (MAG; monoglycerides) are the
most widely used emulsifiers in the food, pharmaceutic,
and cosmetic industries. Beside bulk applications in the
food and dairy industry,38*39 some other applications for
special MAG have been described (Table 1). Li and Ward
reported that MAG containing n-3-polyunsaturated fatty ac-
ids such as eicosapentaenoic acid (EPA) and docosahex-
aenoic acid (DHA) may have positive preventive effects on
Address reprint requests to Dr. Uwe T. Bomscheuer,
nische Biochemie, Universitlt Stuttgart, Allmandring
gart, Germany
Received 15 August 1994; accepted 24 October 1994
Institut fiir Tech-
31, 70569 Stutt-
tadecanoylglycerol is used as a hair care additive.4’
Monoacylglycerols are manufactured on an industrial
scale by continuous chemical glycerolysis of fats and oils at
high temperatures (220-250°C) employing inorganic alka-
line catalysts under a nitrogen gas atmosphere. The product
produced by this strategy has several drawbacks (e.g., low
yield, dark color, burnt taste).39 Molecular distillation is
necessary because MAG need to be highly pure in the food
industry, since they have better emulsifying properties than
a mixture of different acylglycerols.24 The chemical syn-
thesis of regioisomerically or enantiomerically pure MAG is
based on the esterification of protected glycerols with acid
chlorides and subsequent hydrolysis of the keta1.4246
In the last decade, many approaches have been investi-
gated in the enzymatic synthesis of MAG. These are selec-
tive hydrolysis using 1,3-regiospecific lipases, esterifica-
tion of fatty acids or transesterification of fatty esters with
glycerol, and the glycerolysis of fats or oils (Scheme I).
Many different reaction systems for batch or continuous
production have been used, and several aspects such as
type, purity, and immobilization of the biocatalyst, temper-
atures, and solvent system have been investigated in detail.
This review presents, compares, and discusses the state
of the art for the enzymatic synthesis of MAG.
Hydrolysis
The continuous hydrolysis of fats and oils to produce fatty
acids and glycerol has been a standard large-scale process in
industry. The fats are hydrolyzed by countercurrent treat-
ment with steam at high temperatures and pressures.39*47
Enzymatic hydrolysis using nonspecific lipases offers an

Enzyme and Microbial Technology 17:578-586, 1995

0 1995 by Elsevier Science Inc. 0141-0229/95/$10.00
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 Lipase syntheses of monoacylglycerols: U. T. Bornscheuer
Table 1 Application of monoacylglycerol The best results were obtained at pH 7.5 with 23% MAG
(mainly monoricinolein) and 66% ricinoleic acid after 3 h
Application References reaction time. Precipitation of the acid by the addition of
salt raised the MAG content up to 65%. To investigate the
Cosmetics, toothpastes, etc. Baumann et al., 3s Sonntag3g structural properties of the active site of lipase B from
Emulsifier, mainly in food Kotting and EibP5 Pseudomonas fragi 22.39B, glycerides were hydrolyzed by
technology Tsuzuki et al. in a homogeneous solvent system (buffer-
Medicine and therapy Li and Ward, a Akoh et aLg8 tetrahydrofuran). The lipases were made soluble in organic
Hair care additive Weiss4’
solvents and the effect of the chain length and number of
unsaturated bonds were investigated.61
A more efficient preparation of 2-MAG in a biphasic
system was presented by Mazur et a1.62 The system con-
alternative process for the production of fatty acids under sisted of hexane, aliphatic alcohol (e.g., 2-butanol), and an
mild conditions. Several methods have been described in aqueous buffer containing 1,3-specific lipase (lipozyme). It
the literature, including reactions in the presence4&“’ or was found that higher selectivity was induced by the ester-
absence 52*53of organic solvents, and examples for contin- ification of the released free fatty acids with the aliphatic
uous hydrolysis employing different reactor systems (e.g., alcohol (namely primary alcohols) in a second step. The
hollow-fiber bioreactors). 54-57The latter examples were re- yield was 70%, and the method was applicable for triglyc-
viewed by Mukherjee.37 erides from C,:, to C,4:0.62 Another possibility for the in-
To obtain MAG by enzymatic hydrolysis, it is necessary duction of selectivity was achieved by the hydrolysis of fats
to control the reaction so that complete hydrolysis is in oil-rich microemulsions in the presence of anionic sur-
avoided. The easiest way is the application of 1,3-sn- factants. Holmberg and &terberg63 were able to obtain
specific lipases yielding 2-MAG [Scheme I, Eq. (l)]. The 2-MAG in 80% yield under optimum conditions. However,
main problems in this type of reaction are the low overall slow acyl migration was reported and higher yields were
yields of MAG, because 2 mol free fatty acid mol- ’ MAG impossible because of complete hydrolysis to fatty acid and
is produced and acyl migration [from the 2 to the l(3) glycerol.
position of glycerol] has to be suppressed. Partial hydrolysis The problem of acyl migration and the stereo- and re-
was achieved for some seed oils, such as mustard, crambe, gioselectivity of lipases in reactions of glycerol derivatives
and honesty.58,59 Flenker and SpenefiO hydrolyzed castor were investigated by several groups646R and are beyond the
oil by using a 1,3-specific lipase from Rhizopus arrhizus. scope of this article.

OR OR Esterification of fatty acids or esters with glycerol

l.3-sn-speclfic l1pase
. (1)
2 R-COOH The discovery of the high stability of lipases in organic
h n
RO OR HO OH solvents offered the possibility of a reverse reaction of hy-
Fat/Od 2-MAC drolysis, esterification using free fatty acids (FFA), or
transesterification using esters [Scheme 1, (Eqs. 2 and 3)].
For that purpose conditions are needed, in which the enzyme
OH(R) will catalyze the synthesis reaction rather than the hydroly-
sis. Most important, low water content and low water ac-
H& OR tivity are necessary. 69-71To achieve a high-yield formation
Fatty sad I(2)-MAC of 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 the
OH(R) addition of molecular sieves’*,‘” or reactions under reduced
R~oxR
IlpW
(3) pressure.74 Examples for the synthesis of MAG by esteri-
- K-OH
h fication are listed in Table 3 and are discussed in more
HO OR(H)
detail.
Ester I(2)-MAC

Influence of the fatty-acid chain length

OR OH OH(R)
IlpW In 1977, Tsujisaka et al. 7s demonstrated the versatility of
-
A (4) lipases for the esterification reaction. With lipases from
A
RO OR H& dR8 HI Aspergillus niger or Rhizopus delernar, esterification of
Fat/Od I(2)-MAC oleic acid occurred almost exclusively at the sn-1 or sn-3
positions of glycerol. On the other hand, lipases from Geo-
Scheme 1 Schematic drawing of lipase-catalyzed methods for trichum candidum and Penicillium cyclopium esterified
the synthesis of monoacylglycerol (MAGI using selective hydro- only long-chain fatty acids, and esterification occurred ran-
lysis with 1,3-sn-specific lipases [eq. (I)], esterification of fatty
acids [eq. (2)] or esters [eq. (3)] with glycerol and the glycero-
domly.75 The influence of the fatty-acid chain length was
lysis of fats oils [eq. (4)]. By-products such as diacylglycerol, also studied by other groups. 7G78 Jansisenet al. 76 found that
triacylglycerol, and free fatty acid are not shown, for clarity a higher monoester formation was possible with short-chain

Enzyme Microb. Technol., 1995, vol. 17, July 579

Review
Table 2 Methods to achieve a selective formation of MAG in alcohol were also performed in a continuous two-fixed-bed
esterification reactions segment with dehydration of the reactional medium be-
tween each pass. At a residence time of 1 h between each
Method Principle References pass, 44.7% MAG was formed almost exclusively and a
high stability of the lipase was reported.
Addition of molec- Water removal Yamaguchi and The influence of organic solvents on the synthesis of
ular sieves Mase,72 Gan- oleylglycerols was recently studied by Pastor et a1.80 and
ceF3
Otero et al.“’ The direct esterification of oleic acid with
Reduced pressure Water removal Miller et a/.74
Adsorption of Shift of equilib- Berger and glycerol in n-heptane gave only a low MAG content (max.
glycerol on sup- rium, enhanced Schneider87,89 7.2%) even at high temperatures (max. 70°C). The trans-
port material selectivity esterification of ethyl oleate with glycerol in different sol-
Adsorption of Shift of equilib- Van der Padt et vents led to a higher formation of MAG, especially without
MAG on in-line rium, enhanced alksl the addition of water. Here, at optimized conditions up to
column selectivity
Addition of phe- Enhanced solubil- Steffen et a/.92 68%, MAG were formed after 2 h by lipase from Candida
nylboronic acid ity of glycerol, antarctica in acetone. Furthermore, at short reaction times,
protective agent it was found that the reaction was faster and more selective
Reaction in solid Shift of equilib- Weiss4’ to MAG production when a polar solvent (e.g., acetonitrile)
phase rium
was used.

Reactions in reverse micelles

fatty acids in polar solvents, whereas a higher di- and tri-
ester synthesis took place with longer-chain fatty acids in
nonpolar organic solvents. The ester molar fractions at equi-
librium were found to depend on the fatty chain length in
the absence of a solvent. A computer program (Two-phase
Reaction Equilibrium Prediction) was used to predict equi-
librium amounts of fatty acids and esters formed.
A high-yield synthesis of MAG was achieved by the
addition of molecular sieves.72 Under optimal conditions
the conversion of oleic acid was 97.3% and 74 wt% MAG
were formed by a lipase from Penicillium camembertii.
Long-chain unsaturated and medium-chain saturated fatty
acids were efficient acyl donors. The conversion of diacyl-
glycerol (DAG) to MAG was also observed by the addition
of molecular sieves.
Monooleylglycerol as a model MAG
Monooleylglycerol has been used by many research groups
as a model MAG. Surprisingly, the achieved conversions
and selectivities differed significantly (Table 3) depending
on 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 of
monooleylglycerol were achieved compared to reactions
catalyzed by lipozyme, in which dioleyl- and trioleylglyc-
erol were the most abundant products. They also investigated
esterification with [l-‘4C]-labeled oleic acid with mono-
and dioleylglycerol and the hydrolysis of [ 1-14C]-labeled
trioleylglycerol.
The formation of MAG in tertioamylic alcohol, trichlo-
rotrifluoroethane, and supercritical carbon dioxide by
mycelium lipase from R. arrhizus was reported by Gan-
cet.73 Whereas in tertioamylic alcohol no formation of
DAG was observed (17.9% MAG), in trichlorotrifluoro-
ethane almost similar amounts of MAG (37.10/o), DAG
(30.5%), and oleic acid (32.4%) were detected. The highest
MAG content (44.1%) was achieved in supercritical carbon
dioxide at 77 bar and 32°C.73 The reactions in tertioamylic
The examples explained above were performed in solvent-
free systems or in the presence of organic solvents. As
alternative reaction systems, reverse micelles were also
used for the lipase-catalyzed formation of MAG as reaction
media.82-86 Besides the advantages of a high interfacial
area, easy preparation, and good enzyme-substrate contact,
the encapsulation of glycerol into the reverse micelles over-
comes the problem of the low solubility of glycerol in or-
ganic solvents.
Hayes and Gularis4 employed a 1,3-specific lipase from
R. delemar, but mainly l-MAG was formed. This observa-
tion was related to an acyl migration of a fatty-acid residue
from the 2- to the l(3)-position of glycerol. The conversion
reached S&60%, and the reaction rate decreased in the
order: oleic acid > caprylic acid > myristic acid = lauric
acid > stearic acid = palmitic acid. A higher conversion
(80-90%) was reported by Fletcher et a1.,82 but the lipase
employed was not 1,3-positional-specific and both l- and
2-MAG were formed. This was also reported by Bom-
scheuer et al. 85 for the reaction between glycerol and lauric
acid using purified lipase from Pseudomonas cepacia in
AOT (Dioctylsodiumsulfoccinate) microemulsions.
Whereas lipase from Chromobacterium viscosum was found
very stable in AOT reverse micelles,** R. delemar lipase
showed only poor stability in the same system.84
Systems based on selective adsorption
The high-yield synthesis of regioisomerically pure 1(3)-
MAG in organic solvents on a multigram scale was reported
by 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 glycerol
onto a solid support (e.g., silica gel, charcoal, celite) 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-
selective lipase (e.g., R. delemar, Rhizomucor miehei,
Chromobacterium viscosum) were added, thus mimicking
an artificial liquid-liquid interphase. After the reaction was

580 Enzyme Microb. Technol., 1995, vol. 17, July

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

Lipase Substrate” Main product0 References

Mucor miehei OA l-MAG (max. 32%), Yamane et al.,93 Hoq et al.94

80% conv.
Aspergillus niger, Rhizopus OA l,(3)-MAG or random Tsujisaka et al.75
delemar, Geotrichum candi-
dum, Penicillium cyclopium
Penicillium camembertii OA MAG (max. 74% wt) Yamaguchi and Mase7*
Penicillium sp., Lipozyme OA Low to high MAG Schuch ahd Mukherjee79
Rhizopus arrhizus OA MAG (17.9-44.1%) Gancet73
Candida antarctica OA, ethyloleate MAG (7.268%) Pastor et al.*O
Rhisopus delemar OA and others l-MAG, 50-60% conv. Hayes and Gularis4
Lipozyme OA, SA MAG, 30-70% conv. Singh et al.86
Lipozyme eg., (S)-17-hydroxystearic acid MAG (max. 84%) Steffen et al.92
Geotrichum candidum, EPA, DHA 6.4-65% MAG Li and Ward,40 Akoh et al.,g6
Pseudomonas sp.. Mucor Osada et aLg7
miehei
Penicillium cyclopium, Rhizo- Solid FAA (e.g., C,,) MAG (max 96%) Weiss?’
pus sp.
Chromobacterium viscosum G-G,, cl*:, High MAG Janssen et al.76
Humicola lanuginosa G-Go MAG Ibrahim”
Aspergillus niger c4-c,,, h3:,. %3:* MAG Tahoun et a/.‘s
Rhizopus delemar, Mucor mie- e.g., C&,-acids l,(3)-MAG, >70% Berger and Schneidera7,sg
hei, Chromobacterium visco- (sat. and unsat.) yield
sum
Candida rugosa Caprylic acid MAG >90% 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., maximum; conv,. conversion

completed, both the enzyme and the solid support were
removed simultaneously by simple filtration while the es-
terification products remained in solution. Under optimized
conditions, the MAG content achieved 70%, but higher
yields were found to be impossible, because at high con-
centrations, l(3)-MAG served as a better substrate than
glycerol. 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.87
The successof the method of Berger and Schneiders’ was
“Recychng
W petitively adsorbed compounds such as DAG and free fatty
OH i OH
bpt3.X
+ I eq. acyl donor _ + “Glycerides”
HO
h
OH
Glycerol
(adsorbed1
demonstrated by using numerous acyl donors, such as free
fatty acids (from C, to C,, in saturated and unsaturated
conditions), fatty-acid methyl esters, vinyl esters, synthetic
triacylglycerols, and natural fats and oils. Various solid
monoacylglycerols 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 adsorption
column.91 In this reactor, an internal oil circuit (containing
the fatty acid and produced glycerides)i and an external glyc-
erol-water circuit are kept separated using a hollow-fiber
membrane module. The lipase is adsorbed at the inner fiber
side and an in-line column is placed in the oil phase circuit
of the reactor. Van der Padt et ~1.~’ $ound that monoacyl-
glycerols (e.g., monocaprinate) adsorbed preferentially on
the in-line column, thus removing MAG from the reaction
mixture, shifting the equilibrium and repressing the further
esterification to DAG and TAG. A high purification factor
(>90%) was achieved by mild (off-line) elution from com-
I acids. The authors implied that the membrane reactor can be
used in a continuous process using a sequence of columns.
HO
h
OAcyl
They estimated a production of 60 mol (15 kg) monoester
g ~ ’ enzyme. The half-life time of the lipase from Candida
rugosa was given as 50 days.

1 (3).m-monoacylglycerol
Other systems
Scheme 2 Synthesis of regioisomerically pure l(3)-mono-
acylglycerol (MAGI using adsorbed glycerol; isolation and recy- The additive, phenylboronic acid (PI&A), was described as
cling of by-products (according to Berger and Schneider3? a solubilizing and protecting agent in the esterification of

Enzyme Microb. Technol., 1995, vol. 17, July 581

Review
glycerol with uncommon fatty acids in the presence of or-
ganic solvents.92 The highest yield of MAG (84%) was
obtained using (S)-17-hydroxystearic acid as the substrate
and lipozyme as the biocatalyst in n-hexane. Steffen et a1.92
found that MAG were produced exclusively at a high ratio
of free fatty acid (PPA):glycerol:PBA (e.g., 1:5:5); 12-
hydroxy-stearic acid, 9(10)-acetonyl-stearic acid, and
9( 10)dimethylmalonylstearic acid served as further sub-
strates .
Another method was based on the enzymatic esterifica-
tion of glycerol with solid fatty acids and their methyl esters
employing lipases from Penicillium cyclopium or Rhizopus
SP.~~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 of
monopentadecanoylglycerol, a hair care additive. Under
optimized conditions almost quantitative conversion of the
PFA to the corresponding MAG (96%) was achieved. The
selectivity 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 identical reaction conditions, lipase
from Rhizopus sp. yielded only 5-8% MAG, and DAG was
the main product. Disadvantages of the process described
by Weiss41 were the thermal inactivation of the lipases,
which took place in the downstream process, and the large
excess of glycerol.
The continuous production of glycerides from liquid
fatty acids and glycerol by lipase from Mucor miehei in a
microporous, hydrophobic membrane reactor was reported
by Yamane et a1.93 and Hoq et a1.94 Thirty-two percent
MAG (mainly l-MAG) was produced at 80% conversion.
The half-life of the enzyme was extrapolated to 54 days.94
The application of membrane bioreactors for the esterifica-
tion of fatty acids with glycerol and the hydrolysis of oils
has been comprehensively reviewed by Kloosterman et
a1.95
Of special interest for pharmaceutic application are n-3
polyunsaturated fatty acids such as eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) (Table I). EPA
and DHA are only completely adsorbed as glycerides such
as monoacylglycerols.40 Their synthesis was performed by
esterification with lipases from Geotrichum candidum,96-97
Pseudomonas sp . , or M. miehei.40 The reaction systems
were optimized with respect to water and glycerol content,
organic solvent, temperature, and other conditions. The
content of MAG varied widely, from 34.5% MAG (mainly
EPA and DHA-MAG40) to 65% EPA yield96 and 6.4%
(DHA-MAG) to 8.65% (EPA-MAG).97
1.2-O-protected glycerol fatty acid ester
Scheme 3 Synthesis of monoacylglycerol (MAG)
582 Enzyme Microb. Technol., 1995, vol. 17, July
Synthesis of MAG using protected glycerol
To overcome the problem of subsequent acylation, which
may occur in esterifications with pure glycerol, several re-
search groups employed protected glycerols. This also of-
fered an advantage for the synthesis of position-specific
and/or enantiomerically pure, or at least enriched, MAG.
Usually, a fatty acid was enzymatically esterified with a
derivatized glycerol moiety that had two of the three hy-
droxyl positions blocked (Scheme 3). Once esterified, the
blocking group then was removed in a subsequent step,
resulting in the formation of regioisomerically pure MAG.
As discussed in the previous section, one problem was
the removal of water generated during esterification. This
was achieved by using open test tubes,98 reactions under
reduced pressure,99 or by the addition of molecular
sieves. loo By controlling substrate ratio, type of acyl donor,
cleavage catalyst, and concentration, it was possible to syn-
thesize MAG in a purity of up to 100%.
Several reports have dealt with the esterification of oleic
acid with 1,2-0-rat-isopropylidene-sn-glycerol (1,2-0-
IPG; 2,2-dimethyl-1,3-dioxolane-4-methanol) catalyzed by
immobilized lipase from M. miehei (lipozyme).98-*00 Al-
though the authors investigated the same system, the
achieved conversions, yields, and optimum temperatures
differed greatly. The reason may be the use of different
amounts of lipozyme, substrate ratios, and reaction times.
Pecnik and Knez99 reported a strong influence of pressure
and temperature. Optimum conditions were found at 0.057
bar and 55”C, resulting in 80% conversion of oleic acid
after 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 interval
from 10-70°C.99 Omar et al. loo reported the highest con-
versions (>90%) at an oleic acid-IPG ratio >3. loo The
half-life of lipozyme was calculated to 20 days, and the
productivity was 869 g product g-’ lipase. The specificity
of lipozyme toward other fatty acids with chain lengths
from C,, to C,, and C1sL2to C,,:, were found to be similar
to those found for oleic acid (C,,:,). HeB et al.lol synthe-
sized l-monostearoylglyceride from either stearic acid or
ethyl stearate with 1,2-0-IPG and lipase from Pseudomonas
cepacia followed by hydrolysis of the acetonides with boric
acid. The highest reaction rates were found in n-heptane or
n-dodecane; the maximum conversion was 84%. Increasing
the alkyl chain length of stearate led to a decrease in the
reaction rates. The remaining activity of the lipase was very
high, up to 64% after 24 days’ incubation time was deter-
mined. The authors also observed an enantiomeric excess
k selective hydrolysis Al
;x;p O _ J * Ho)+
R HO
1.2-O-protected-glycerol-fatty ester 3.MAG
from protected glycerols and esters followed by cleavage of the protective group
 Lipase syntheses
(%ee) of 11 for 1-monostearoylglycerols synthesized from
ethyl stearate and 1,2-0-IPG.
Another approach in the synthesis of MAG with IPG was
based on the transesterification employing either meth-
yloleate in n-hexaneg8 or the application of enol ester. ‘“2*‘03
In the former case, the conversion ranged from 46.9-67.8%
depending on the lipase type and amount of solvent. After
acid catalyzed cleavage of the protecting group, between
27.1 and 50.3% MAG was determined. Another attempt
was based on the application of enol esters, which offer the
advantage of a shift of the reaction equilibrium due to irre-
versible tautomerization of the by-product vinyl 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 corresponding
acetonides in short reaction times (up to 24 h) in a solvent-
free system or in the presence of n-pentane. Wang et al.
produced glycerol monovalerate and glycerol monoacetate
using the same process. Moreover, these monoacylglycer-
01s have been enantiomerically enriched, and up to 67%ee
was achieved. In this reaction, which was also catalyzed by
lipase from P. cepacia, the unreacted IPG was almost enan-
tiomerically pure (92%ee). lo3
An important step in the reaction employing blocked
glycerols is the cleavage of the protecting group. Various
methods have been investigated, and ranged from mild hy-
drolysis employing boric acid’” in 2-methoxyethanol or
iodine in methanollo to mean and strong acidic conditions
using concentrated HCl or trifluoroacetic acid.98,99.‘02 Un-
fortunately, strong acidic conditions resulted in the interes-
terification between two MAG molecules, leading to the
formation of DAG and glycerol as by-products and acyl
migration.98.102
The influence of the protecting group on the reaction rate
and optical purity was investigated for the enzymatic hy-
drolysis of different 1,2-ketals of glycerol.106 The highest
selectivity was found for the hydrolysis of 2,2-dimethyl-
1,3-dioxolane-4-methanol butanoate yielding glycerol
monobutanoate and catalyzed by proteinase from Aspergil-
lus oryzae. The umeacted ester had an enantiomeric excess
of 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 reaction
rate 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 only
poor results. lo6
Glycerolysis
As outlined previously, the glycerolysis of fats and oils
employing alkaline catalyst represents the process currently
used in industry for the large-scale synthesis of MAG.
Enzymatic glycerolysis offers the advantage of a high
space-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 of
MAG.
Initiated by the work of Yamane et al., lo7 lipase-
of monoacylglycerols: U. T. Bornscheuer
catalyzed glycerolysis received considerable attention, and
several papers dealt with the glycerolysis reaction of model
fats and a wide variety of natural fats and oils. The reactions
were performed in reverse micelles,108~‘09 in the pres-
ence”O. ’ l1 or absence’ ‘* of organic solvents, and in a sol-
vent-free-solid-state system.‘13-’ I6 Problems employing
the hydrophilic substrate glycerol in organic solvents and
analytical problems for the quantitative determination of
partial glycerides were described by Ferreira-Dias and Fon-
seca. “’
Yamane et u1.‘07 studied the glycerolysis reaction of
corn oil by Pseudomonusfluorescens lipase in a batch sys-
tem without the addition of surfactants or emulsifiers. At
40°C and 3.7% water content in the glycerol phase, 20.4%
MAG content was determined. It was also reported that
lipase was inhibited and inactivated by fat peroxide in the
course of batch and continuous glycerolyses of safflower
oi1.‘18 Other authors”3m”6,“R improved their system by
carrying out the reactions first in a liquid-liquid emulsion
state, then cooled until the glyceride-glycerol mixture be-
came solid. The strong increase in the formation of MAG
after reaching solid-state was related to the crystallization of
MAG from the reaction mixture. This led to a shift in the
reaction equilibrium. Using this method, a high (70-99%)
formation of MAG from various natural fats and oils such as
beef 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 was
the reaction temperature, and much care was taken for the
determination of optimal temperature programming. The
authors created the term “critical temperature” (Tc) for the
sharp transition temperature between the high and low
monoglyceride states. Further optimization of the reaction
and 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 and
stabilities of lipases from various sources were recently
studied using triolein as a model triacylglycerol. It was
found that bacterial lipases were very stable, whereas yeast
and mold lipases were quickly inactivated.“s A further in-
crease of the operational stability was achieved by immo-
bilization of lipase on Celite. With lipase from Chromobuc-
terium viscosum 99% MAG content was achieved after in-
cubation for 120 h at 8°C (first 8 h at 25°C).
A correlation between fat composition and MAG yield
by means of chain length and saturation profile of the fatty
acids was found in the solid-state glycerolysis of palm oil-
based fats and blends with rapeseed oil. The detailed com-
positional analysis of the reaction products strongly indi-
cated the preferential crystallization of MAG, ’ 19,l *O espe-
cially for the accumulation of saturated fatty acids such as
C,,:, in the MAG fraction. Optimal conditions were found
at a saturated fatty-acid content of 20180% of the original
fat, whereas at a content >80%, the formation of DAG was
favored. McNeil1 and Berger’19 claimed that the yield of
MAG from a reaction carried out below the Tc can be
predicted from the fatty-acid composiition of the substrate
using simple linear regression analysi,s.
Stevenson et al. l*l reported the glycerolysis of tallow in
solid-state with the immobilized lipase lipozyme in a batch
and a continuous system. The yield of MAG was 35% in the
Enzyme Microb. Technol., 1995, vol. 17, July 583
Review
(solid-state) batch system. For continuous production, the was used. Whereas hydrolysis gives only unlabeled MAG,
liquid reaction mixture was pumped through a column filled glycerolysis should lead to the formation of 2 mol of labeled
with lipozyme, resulting in a decrease in the MAG yield, and 1 mol of unlabeled MAG mol - ’ of triglyceride. The
because crystallization of MAG had to be avoided. An in- rate of glycerolysis was found to approximate that of hy-
crease in the overall MAG yield up to 69% was achieved by drolysis. Regarding the second assumption (acyl migra-
extraction of MAG into n-hexane, filtration of the enzyme, tion), 2-MAG should give I-MAG, which is regarded as a
and recycling of the nonmonoglyceride portion with fresh substrate for a subsequent hydrolysis reaction, also reducing
tallow in another reaction. the final MAG yield. From four 1,3-specific lipases tested,
Other systems for continuous glycerolysis were per- those from Rhizopus sp. gave almost similar results,
formed using a membrane bioreactor’22 or lipase immobi- whereas lipase from porcine pancreas was less active. lo9
lized on liposome in reverse micelles. lo8 In the microporous
hydrophobic membrane bioreactor, edible oil was glycero-
lyzed by lipase from P. fluorescens, resulting in the forma- Conclusions
tion of 5-25% MAG. The half-life of the enzyme was 3 The examples shown in this review demonstrate the diver-
weeks at 40°C.122 Chang et ~1.~~~employed a continuous sity of lipases in a wide variety of reaction systems that have
stirred-vessel bioreactor with polysulfone membrane for the been used for the synthesis of an apparently simple reaction,
glycerolysis of olive oil in AOT/iso-octane reverse micelles the production of monoacylglycerides. Although so far no
using immobilized lipase from C. viscosum. The lipase was systems presented here have been commercialized on an
immobilized on liposome (small unilamellar vesicle) as a industrial scale, the chances for a lipase-catalyzed synthesis
matrix. The polysulfone membrane was 12.5 km thick, and of emulsifiers such as MAG are increasing, for several rea-
the pore diameter was a maximum of 25 nm. After a l-day sons. First, according to Fu1ch,‘23 “the progress in genetics
delay, a steady state was achieved, resulting in 80% con- and in process technology enables the enzyme industry to
version of olive oil during 6 days of operation. The highest offer products with improved properties and often at re-
productivity of 1-monoolein (90 pmol h- ’ ml - ’ outlet) duced costs. ” Second, some newly developed high-yield
was obtained at 2.0% (v/v) of initial substrate concentra- formations of MAG shown here may become more com-
tion. Optimum flow rate of olive oil was 2.5 ml hh’, and petitive in standard chemical processes. Lower energy con-
the water content was below 8% (w/v). The operational sumption, mild conditions, higher space-time yields and
stability of the liposome-lipase was extrapolated to be 45 selectivities, and better product quality should be men-
days, whereas the half-life of the free enzyme in reverse tioned. In addition, the demands for “green technologies”
micelles was only about 4 days. In the latter example, the and “consumer-friendly” products are increasing, and bet-
activity of the immobilized enzyme was only slightly lower ter acceptance should be possible.
compared to the free biocatalyst.‘08 A two-fixed-bed seg- On the other hand, a prediction of lipase-catalyzed reac-
ment for the continuous glycerolysis of beef tallow was tions with respect to substrate specifity, time, and yield, for
developed by Gancet .73 At a residence time of 75 min, a example, is still difficult. Tedious and skillful experiments
yield of 38.8 g MAG 100 g-’ beef tallow was reported. seem to be necessary for the development and optimization
The reactor system was run for several months with a rather of a new process or method. From an optimistic point of
good operational stability of the mycelial lipase from R. view, these problems may be reduced in the near future as
arrhizus. a result of new developments and further knowledge of the
Another high-yield formation of MAG was based on the mechanisms and actions of lipase under natural and unnat-
presence of a secondary or tertiary alcohol in the reaction ural conditions.
mixture 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 at Acknowledgment
7O”C, 82-90% MAG was synthesized by the immobilized The author thanks Prof. T. Scheper for discussion and re-
thermostable lipase from Candidu antarctica. The system
vision of the manuscript.
was also applied for esterification and transesterification
reactions using fatty acids and their derivatives from C, to
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