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ReviewEnzymes as biocatalysts in the modification ofnatural lipidsFrank D Gunstone*Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland
Abstract: Though designed by nature to effect hydrolysis of lipids, lipases can, under appropriate
reaction conditions, promote ester formation through reaction of acids and alcohols (esteri®cation) or
of esters with acids (acidolysis), alcohols (alcoholysis), or other esters (interesteri®cation). Compared
with chemical processes already carried out on an industrial scale enzymic reactions occur under
milder (and `greener') conditions though they may take longer. Of greater signi®cance is the speci®city
shown by the enzymes which permits the formation of lipid derivatives not easily prepared by
conventional laboratory procedures.
This review describes the lipases and their various speci®cities and reports on their use in hydrolysis
and in the production of phospholipids, fatty acids, alkyl esters, mono- and di-acylglycerols, tri-
acylglycerols, other esters, and amides. Some of these have already led to marketable products but for
the most part the full potential of these reactions has yet to be realised. The reactions of other enzymes
promoting interesting reactions at unsaturated centres are also described.
# 1999 Society of Chemical Industry
Keywords: lipases; enzyme-catalysed hydrolysis; esteri®cation; acidolysis; alcoholysis; interesteri®cation;polyunsaturated fatty acids; medium-chain triacylglycerols; mono-, di- and tri-acylglycerols; wax esters; sugaresters; amides; enzyme-catalysed reactions; ole®nic centres
1 INTRODUCTIONCommercial oils and fats are now produced at levels
around 100 million tonnes per annum and are used
mainly for food (80%), animal feed (6%), and for
oleochemical purposes (14%) the great majority
(90%) of which is for the production of soaps and
other surface-active materials. On this basis it is not
surprising that much research in the lipid ®eld is
devoted to nutrition which also drives many of the
processing techniques. The natural materials are not
always best suited for the nutritional demand made on
them and for the whole of the 20th-century (and
before) lipid scientists and technologists have been
engaged in improving quality in nutritional terms by
blending, fractionation, partial hydrogenation, and
interesteri®cation. Some of these developments have
brought their own problems as in the recognition that
acids with trans con®guration, formed during partial
hydrogenation, as well as saturated acids are not
nutritionally desirable beyond very modest levels.
During this century partial hydrogenation, fractiona-
tion, and interesteri®cation have been developed and
used extensively to modify lipids. Despite, or because
of, their failings these processes are being improved
continually by the makers of equipment and catalysts.
Other drivers come from the growing demand for
`natural' foods and from environmental concerns to
reduce energy requirements and to minimise waste as
well as economic pressures to produce good products
at acceptable prices.
Another area of R and D is the production of `better'
natural products, ie lipids with a more desirable fatty
acid and triacylglycerol composition. These are being
developed in a number of ways which were described
in a recent review.1
. The domestication of wild plants (eg cuphea,
calendula, etc) often to produce high levels of a
single acid for oleochemical purposes.. Modi®cation of existing oilseed plants by con-
ventional seed breeding as in the development of
double zero rape seed from high-erucic rape seed
oil or by genetic modi®cation. So far the latter
have been directed mainly to the production of
species which show agricultural traits such as
herbicide resistance and pesticide resistance, etc.
Journal of the Science of Food and Agriculture J Sci Food Agric 79:1535±1549 (1999)
* Correspondence to: Frank D Gunstone, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, ScotlandE-mail: fgunst@scri.sari.ac.uk(Received 1 March 1999; accepted 18 May 1999)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1535
Only recently have materials with changed fatty
acid composition begun to appear. The ®rst
example is a high-laurate canola but other
interesting materials are following quickly.. Production of oils from microbial sources such as
oils rich in arachidonic acid (Mortierella alpina)and in docosahexaenoic acid (Crypthecodiniumcohnii).
This review is devoted to a different area of lipid
research viz the use of biocatalysts in general and
lipases in particular to effect changes in the lipid
mixtures produced naturally. The lipases have long
been known as important enzymes in lipid metab-
olism, promoting complete or partial hydrolysis of
glycerol esters. Attempts have been made to exploit
these as hydrolysis catalysts and the following quota-
tion is taken from a book published in 19272 at the end
of a two-page description of technical hydrolysis with a
preparation from castor seeds. `The lipase fat-splitting
process is a step in the right direction in that it is an
attempt to use Nature's active chemical agents ± the
enzymes which by operating at natural temperatures
avoid the unnecessary expenses of fuel for steam and of
manufactured chemicals as reagents'. Those prescient
word were penned three-quarters of a century ago. In
the intervening years steps in this direction have been
slow and faltering but now they are gathering pace. In
the last ten years lipase-catalysed reactions have been a
subject of increasing interest and growing importance
resulting from the realisation that, under appropriate
conditions, the biocatalysts can promote ester forma-
tion as well as ester hydrolysis and that, with careful
choice of lipase, reaction conditions and substrates, it
is possible to control acylation and deacylation to
produce speci®c fatty acids and triacylglycerols. This
ability has developed at the same time as a demand for
speci®cally structured molecules not readily available
from natural sources.
There is much research activity in this ®eld. Con-
ferences on lipase reactions are frequent and popular.
Many interesting molecules and mixtures have been
produced but only a few have led to commercial
products.1 The increased cost of using enzymes means
that so far only low-volume, high-value products have
been produced in this way. Nevertheless the wide
interest in this topic, in both academic and industrial
laboratories, suggests that further products will soon
come to the marketplace.
2 LIPASESCommercial lipases are available from animal, plant,
and microbial sources including those listed in Table
1.
Though designed for hydrolysis of lipids and related
Table 1. Selectivity shown by lipases fromselected sources
Lipase source
Glycerol
reactivity Discrimination against
Reaction
con®ned to
Aspergillus niger 1,3 �2
Aspergillus sp None
Candida rugosaa None D4, 5, and 6 acids/esters
C antarcticab None D4, 5, and 6 acids/esters
C lypolytica 1,3>2
C parapsilosis
Chromobacterium viscosum
Geotrichum candidum D9c acids
Humicola lanuginosa 1,3 �2
Mucor javanicus 1,3>2
Mucor mieheic 1,3>2 D4, 5, and 6 acids/esters
Oats (Avena sativa) D9 acids
Pancreatic (porcine) 1,3>2
Pre-gastric esterase 1,3>2
Penicillium sp 1,3>2
Phycomyces nites
Pseudomonas cepacia
Pseudomonas ¯uorescens None
Pseudomonas spp
Rape seedlings
Rhizomucor mieheic D4, 5, and 6 acids/esters
Rhizopus arrhizus 1,3>2
Rhizopus delemar 1,3 �2 D4, 5, and 6 acids/esters
Rhizopus japonica
Rhizopus javanicus 1,3>2
a Previously known as Candida cylindracea.b Also referred to as Novozyme 435TM.c Alternative names for the same lipase±also referred to as LypozymeTM.
1536 J Sci Food Agric 79:1535±1549 (1999)
FD Gunstone
molecules, lipases can also catalyse other reactions
associated with acyl groups. These include:
. Hydrolysis: reaction of ester with water producing
acid and alcohol and with hydrogen peroxide to
give peroxy acids.. Esteri®cation: the reverse of hydrolysis ± produc-
tion of ester from acid and alcohol.. Alcoholysis: reaction of an ester with a mono-
hydric alcohol such as ethanol, butanol, lauric
alcohol, or a polyhydric alcohol such as glycerol to
produce an ester with a different alkyl group.
Similar reaction with amines leads to amides.. Acidolysis: reaction of an ester with an acid
leading to exchange of acyl groups.. Interesteri®cation: reaction of one ester with
another leading to scrambling (randomisation)
of acyl and alcohol moieties.
These processes can be selected by choosing appro-
priate substrates and reaction conditions. Increasing
numbers of research papers utilise lipases as bio-
catalysts and cover a range of reactions. Perhaps the
most signi®cant are those aiming to produce triacyl-
glycerols of de®ned composition, mainly on the basis
of their nutritional properties (see Section 8).
Lipase-catalysed processes have attracted attention
because of the mild reaction conditions under which
they occur and the selectivity displayed by these
catalysts. In both these respects they differ from
typical chemical reactions.
Because enzymic reactions occur under mild con-
ditions of temperature and pH and at normal pressure,
they generally require less energy and are conducted in
equipment of lower capital cost than many other
chemical processes. Also, under these milder condi-
tions, the products are purer and less degraded
through alternative high-temperature reactions, so
they are more easily puri®ed and waste disposal is less
of a problem.
Because of the selectivity of many lipases it is
possible to obtain products which are dif®cult to make
by more conventional chemical reactions. These
enzymes may show one or more of several types of
selectivity (Table 1):
. Some distinguish between acids of different chain
length, reacting differently with short, medium
and long-chain acids.
. Reactivity is also in¯uenced by structural features
in the alkyl group of the fatty acid chain such as
double bond con®guration and position with
respect to the acyl function, and the presence
and position of other functional groups such as
branched methyl.. Reaction may differ with the nature of the acyl
source±whether it is free acid, alkyl ester, or
glycerol ester.. In esters of polyols (such as glycerol esters) there
may be a distinction between acyl chains linked to
primary and secondary hydroxyl functions (ie
those in the sn-1/3 and the sn-2 positions) and in
the relative reactivities of monoacylglycerols,
diacylglycerols, and triacylglycerols.
It is likely that speci®city will be further developed with
a fuller understanding of the optimum reaction
conditions (temperature, pH, molar ratio of reactants)
and with better enzymes resulting from modi®cation of
existing lipases or from new sources. This is an area of
active research.
The molecular basis for lipase activity and speci®city
is poorly understood but recent advances in this topic
have been reported for the lipases from Geotrichumcandidum and Candida rugosa,3 Pseudomonas cepacia,4
Rhizopus delemar,5 and for pancreatic lipase.6 It is
believed that lipase activity is associated with a serine
residue in the protein chain. Binding sites, associated
with speci®city, include a hydrophobic cleft or an
L-shaped hydrophobic tunnel approximately 20
carbon atoms long. Phycomyces nites after N-acylation
(C18±C22) of its lysine residue is 40 times more
reactive and has modi®ed selectivity in transesteri®ca-
tion reactions.7 Lipase activity can also be modi®ed by
the presence of surfactants as reported for Rhizopusjaponicus and sorbitan monostearate.8
To demonstrate the speci®city of the lipases from
Rhizopus delemar and Rhizomucor miehei, Sonnet and
Sazzillo9 examined the partial hydrolysis of the
triacylglycerol POS and compared the results with
those obtained with the pancreatic lipase used
analytically on the basis of its 1,3-speci®city (Table
2). Complete speci®city should lead to no oleic acid in
the liberated acids though this value must increase if
hydrolysis proceeds beyond 67%. Signi®cant selectiv-
ity of other kinds has also been reported for Geotrichumcandidum10,11 and for Penicillium sp.12 Many enzymes
Table 2. Demonstration of 1,3-selectivity inthe partial hydrolysis of POS
Glycerol esters Free acid
Lipase source Hydrolysis (%) P (%) O (%) S (%) P (%) O (%) S (%)
Porcine pancreas 39 27 48 24 43 13 44
Rhizopus delemar 51 21 60 18 45 3 52
Rhizomucor miehei 78 19 65 10 36 24 40
P=palmitic
O=Oleic
S=Stearic.
J Sci Food Agric 79:1535±1549 (1999) 1537
Enzymes as biocatalysts in the modi®cation of lipids
discriminate against acids with D4, D5, and D6
unsaturation as is fully demonstrated in Section 5.
Speci®city usually refers to hydrolysis of triacylgly-
cerols and is not always the same in esteri®cation
reactions. For example, Osterberg et al13 reported that
lipases from Rhizopus delemar and from other Rhizopussp were unable to promote hydrolysis of tri-g-linolenin
but promoted acidolysis of triolein by g-linolenic acid
when the latter was used in large excess.
New sources of lipase have been identi®ed in
germinating rape seedlings and in oats (Avenasativa).14±16 The latter displays a similar speci®city to
that shown by Geotrichum candidum except that, unlike
this lipase, it promotes reactions with elaidic acid and
its esters (9t-18:1). Hou et al17 and Hou18 have
reported a search for new lipases, several of which
show promise as useful catalysts.
Reaction systems usually consist of lipase (water-
soluble, though sometimes used in an immobilised
form) with its associated water, the reactants, perhaps
a solvent (water and/or organic solvent), and possibly
additional materials such as silica or a molecular sieve.
Reaction conditions must be controlled so that sub-
strates are present at the reactive interface in appro-
priate form and proportion and products are removed
from the interface so that they do not react further nor
inhibit the desired reaction. For example, silica gel is
often added to reactions involving glycerol since this
molecule is so strongly adsorbed on the lipase that
reaction is inhibited. Instead the glycerol is adsorbed
by the silica and made available to the enzyme only in
appropriately restricted amounts.19
Reactions promoted by lipases are generally rever-
sible, leading to an equilibrium. To get high yields of
the desired product it may be necessary to displace this
equilibrium by removing one of the products. Water
and other volatile products may be removed by
operating at a suitable temperature and pressure or
by using a molecular sieve as adsorbent. Other
products can sometimes be removed from the reaction
mixture by crystallisation and it is for this reason that
reaction may be started at one temperature (say 40 or
50°C) and then reduced to a lower temperature (say
10°C). If one product then crystallises from the
reaction mixture the equilibrium is displaced and
more of that product is formed.
3 HYDROLYSISFat splitting is normally effected by a continuous,
high-pressure, uncatalysed, counter-current process at
250°C and 20±60 bar. Under these high-temperature
conditions the products become discoloured and both
the fatty acids and the glycerol may have to be distilled.
Enzymic hydrolysis can be effected under milder
conditions with a wide range of lipases including
Rhizomucor miehei,20 Aspergillus sp,21 Pseudomonas¯uorescens,22 Candida rugosa,20 and Candida cylindra-cea23 acting at �37°C for about 24hours. High-
melting fats can be hydrolysed under these conditions
in the presence of a little iso-octane.24 Partial hydro-
lysis, exploiting the speci®city of lipases which
discriminate against particular acids to concentrate
these acids, has been discussed in Section 5.
4 PHOSPHOLIPIDSPhospholipases A±D provide for the speci®c hydrolysis
of each of the four ester bonds present in phospho-
lipids, and phospholipase D has been used for
transphosphatidylation, ie exchange of the alcohol
moiety attached to the phosphatidic acid residue.
Phospholipase A2 (or other lipases) can be used to
effect exchange of acyl groups.
Na et al25 used phospholipase A2 to prepare phos-
phatidylcholines enriched with n-3 PUFA. With this
enzyme phosphatidylcholine from egg yolk was hydro-
lysed to lysophosphatidylcholine (30°C, 16h) which
was then re-acylated (30°C, 16h) with a polyunsatu-
rated fatty acid (PUFA)-enriched mixture of acids
containing eicosapentaenoic acid (EPA) (41%) and
docosahexaenoic acid (DHA) (30%). The phospha-
tidylcholine ®nally contained 16 and 11% respectively
of these two acids. Using a 90% concentrate of DHA,
phosphatidylcholine with 35% of DHA was prepared.
Three commercially available lipases (Humicolalanuginosa, Rhizopus delemar, and Candida rugosa)
promoted hydrolysis of phosphatidylcholine to the
lyso derivative or to phosphatidic acid in solvents of
appropriate polarity.26 Egg yolk phospholipids
normally contain arachidonic acid (4.9%) and docosa-
hexaenoic acid (3.5%). Hara et al27 made a compara-
tive study of 19 commercially available lipases for the
hydrolysis of phosphatidylcholine to lyso compounds.
Highest reactivity was shown by lipases from Mucorand Rhizopus species. Virtually no hydrolysis occurred
with Candida lipases. When ®sh oil is incorporated into
the feed these values are changed to 1.5 and 10.9%
and after partial hydrolysis with Mucor miehei lipase
they are raised further to levels of 8.8 and 13.7%.28
Acidolysis of phosphatidylcholine with heptadecanoic
acid (Rhizopus arrhizus, 40°C) gave a product with
98% and 1% of this acid at the sn 1 and 2 positions
respectively indicating a successful and selective acid
exchange.29 Mustranta et al30 used Rhizopus mieheilipase (40°C, 24h) and oleic acid to convert di-
myristoylphosphatidylcholine to its 1-oleyl derivative.
Alcoholysis of phospholipids with ethanol, isopro-
panol or butanol under the in¯uence of lipases from
Mucor miehei or Humicola lanuginosa yields lysophos-
pholipids in high yield (22°C, 24h, 98%). By varying
the reaction time useful mixtures of phosphatidylcho-
line and lysophosphatidylcholine were obtained.31
The 1-lysophospholipid is converted to the 2-isomer
by reaction with ammonia vapour at 75°C. In the
presence of Mucor miehei lipase soy phospholipids
undergo alcoholysis with a range of alcohols (C4±C18)
at the sn-1 position (24h, 70±80% conversion) to give
lysophospholipids. There is no speci®city with regard
to the chain length of the alcohol.32 Transesteri®cation
1538 J Sci Food Agric 79:1535±1549 (1999)
FD Gunstone
of soy phospholipids at the sn-1 position occurs with
methyl esters of C10±C18 acids (Mucor miehei) to
furnish phospholipids with capric (8%), lauric (14%),
myristic (16%), or linolenic acid (5% raised to 15%).33
5 FATTY ACIDSThere has been an increasing awareness in recent years
of the nutritional importance of certain fatty acids such
as g-linolenic acid, arachidonic acid, and docosahex-
aenoic acid. These occur naturally but often only at
low levels along with other acids. There is therefore a
demand for preparations with higher levels of these
acids and, at least for research purposes, for prepara-
tions of virtually pure material. While such material
can be produced by conventional physical and
chemical methods of separation these are not always
appropriate if the material is subsequently to be used
for nutritional studies. This problem has been
successfully overcome by exploiting the speci®city of
those enzymes which discriminate against acids having
double bonds at position 4 as in docosahexaenoic acid,
position 5 as in arachidonic acid, or position 6 as in
g-linolenic acid) The concentration or isolation of such
acids is discussed in the following sections.
(i) g-Linolenic acidg-Linolenic acid (GLA) is an important acid used as a
dietary supplement and available from three major
vegetable sources: the seed oils of evening primrose
(8±10%), borage (20±25%), and blackcurrant (15±
17%). These levels of GLA can be raised by enzymic
enhancement and evening primrose and borage oils
with about twice the original GLA levels are commer-
cially available. Laboratory procedures have been
described by Hills et al34 using lipase from rape
seedlings or Mucor miehei and by Foglia and Sonnet35
using Geotrichum candidum lipase. Procedures are
illustrated in the recent study of Shimada et al.36
Borage oil (22% GLA) is hydrolysed (Pseudomonasspecies, 35°C, 24h) and the acids are selectively
esteri®ed with lauric alcohol (Rhizopus delemar, 30°C,
20h). The latter enzyme discriminates against GLA
which therefore concentrates in the unesteri®ed acids.
These ®nally contain 70% GLA (74% recovery). If the
esteri®cation is repeated, 94% GLA is obtained with
68% recovery. Concentration of GLA by selective
deacylation of the oils is also possible but is less
ef®cient. Shimada et al37 showed that selective
hydrolysis of borage oil (22% GLA) gave glycerol
esters with 46% of this acid (C rugosa, 35°C, 15h)
which could hardly be raised above this value. After
removal of free acids, a second hydrolysis raised the
concentration to 54% with 76% recovery. On a larger
scale 7kg of borage oil gave 1.5kg of upgraded oil
(56% GLA) after removal of free acid by molecular
distillation. Starting with an already upgraded borage
oil (10kg, 45% GLA) high quality g-linolenic acid
(2.1kg, 98% pure, 49% recovery) was obtained by
hydrolysis and two separate partial esteri®cations.38
(ii) Arachidonic acidThe single cell oil from Mortierella alpina is now com-
mercially available as a useful triacylglycerol source of
arachidonic acid containing no n-3 acids. Partial
hydrolysis of this oil with an enzyme which discrimi-
nates against arachidonic acid, leads to enrichment of
this acid in the glycerol ester fraction. When oil con-
taining 25% of arachidonic acid is partially hydrolysed
(Candida antarctica, 35°C, 16h, 52% hydrolysis) the
glycerol ester fraction is mainly triacylglycerols with
50% arachidonic acid (95% recovery). If the process is
repeated the level of arachidonic acid rises to 54%
(88% recovery) and 60% (75% recovery).39 (Shimada
and Sugihara et al39). In a later report Shimada et al40
raised the level of arachidonic acid to 75% using the
two-step process already reported for g-linolenic
acid.36 The oil (25% arachidonic acid) is hydrolysed
to free acids (Pseudomonas sp, 40°C, 40h). Subse-
quently these are esteri®ed selectively with lauric
alcohol (Candida rugosa, 30°C, 16h, 55% esteri®ca-
tion) to give residual free acids with 51% arachidonic
acid (92% recovery). This is raised to 63% (78%
recovery) by urea fractionation and to 75% (71%
recovery) by a second esteri®cation.
(iii) Docosahexaenoic acidSeveral procedures for enzymic enhancement of n-3
PUFA, and especially DHA, have been reported. In
general, ®sh oils are subject to selective hydrolysis or
alcoholysis (ethanol or lauryl alcohol) or free acids are
partially esteri®ed (ethanol or butanol). Several
enzymes have given useful results based on the fact
that they discriminate against DHA which therefore
concentrates in the residual substrate.
Selective hydrolysis of seal blubber oil and menha-
den oil (Candida cylindracea, 30±50°C, 46h) gives
glycerol esters enriched in n-3 acids (Table 3).41
McNeill et al42 and Moore et al43 used lipases from
Candida rugosa and Geotrichum candidum to raise levels
of EPA and DHA from 20±30% in several ®sh oils to
35±45% in partial glycerol esters. These can be con-
verted to triacylglycerols by hydrolysis followed by
reaction with glycerol, both reactions being catalysed
by Rhizopus miehei.Shimada et al44 raised DHA levels of tuna ethyl
esters by selective alcoholysis with lauric alcohol from
23 to 50±52% (Rhizomucor miehei or Rhizopus dele-mar). Higher levels were obtained by starting with
already enriched material (45 raised to 74% and 60
raised to 93%). EPA levels are reduced slightly during
Table 3. Enzymic enrichment of polyunsaturated fatty acids in fish oils
Seal blubber oil Menhaden oil
EPA DPA DHA EPA DPA DHA
Original oil 6.4 4.7 7.6 13.2 2.4 10.1
Residual esters 9.8 8.6 24.0 18.5 3.6 17.3
Enrichment 1.5 1.8 3.2 1.4 1.5 1.7
J Sci Food Agric 79:1535±1549 (1999) 1539
Enzymes as biocatalysts in the modi®cation of lipids
this reaction. This result is expected on the basis of
reaction rates relative to those of ethyl oleate (=100):
EPA 13 and 24, DHA 15 and 11 for Rhizomucordelemar and Rhizopus miehei respectively.
Attempts to raise levels of EPA and DHA have been
described by Haraldsson et al45,46 and Breivik et al.47
These are summarised in Table 4. The same authors
have described ways of separating EPA and DHA by
kinetic resolution using Rhizomucor miehei lipase.
(iv) Erucic acidSonnet et al48 concentrated erucic acid from a high-
erucic rape seed oil (HERO) by complete hydrolysis of
the oil (Pseudomonas cepacia) followed by selective
esteri®cation with butanol (Geotrichum candidum).
This latter enzyme discriminates against erucic acid
and its level was raised from 48% in HERO to �85%
in the unesteri®ed acids.
6 Alkyl estersAlkyl esters based on primary (ethyl, butyl) or
secondary (isopropyl, iso-octyl) alcohols can be made
by esteri®cation of free acids or by transesteri®cation
of glycerol (or other) esters using enzymic catalysts.
Such a reaction is frequently one step in a series of
processes for the concentration of individual acids (eg
Refs 44, 46). Methods have been described for
obtaining esters on a kilogram scale and as a step in
upgrading low-quality material such as acid oil or
soapstock.
Ethyl stearate, for example, has been prepared in
high yield from technical stearic acid in 2kg batches
using Lypozyme.49 Esters were also made in high yield
on the 2kg scale by esteri®cation of oleic acid with
butanol and by transesteri®cation of rape seed oil with
iso-octanol using the lipases from Candida rugosa,Chromobacterium viscosum, Pseudomonas ¯uorescens, or
Rhizopus miehei.50
Acid oils containing a mixture of free acids (40±
80%) and glycerol esters (20±50%) can be hydrolysed
(Candida cylindracea lipase, 35°C, 48h) to acids, or the
acid/ester mixture can be converted to alkyl esters (4:0
to 18:0 alcohols) with Mucor miehei lipase.23 Soapstock
(water 45%, acids 10%, glycerol esters 12%, and
phospholipids 9%) can also be upgraded by chemical
hydrolysis followed by reaction with alcohol (methyl,
ethyl, butyl, isobutyl, tert-amyl) or by transesteri®ca-
tion using Lipozyme or Candida antarctica lipase at
42°C.51
Lipase-catalysed transesteri®cation has been recom-
mended for the preparation of methyl or ethyl (Mucormiehei) and isopropyl or isobutyl (Candida antarctica)
esters from tallow or vegetable oils for use as
biodiesel.52 Similar reactions can be carried out using
diesel fuel as solvent to give mixtures of diesel and
biodiesel.53
Deodoriser distillate54 is a valuable by-product of
the re®ning process which contains free acids along
with the more valuable tocopherols and sterols. To
recover the two latter the acids must be removed and
this can be achieved by distillation after enzyme-
catalysed conversion to methyl esters (Randozyme,
50°C) in yields up to 96%.
7 MONO- AND DI-ACYLGLYCEROLSMonoacylglycerols, sometimes with diacylglycerols,
are important food-grade emulsi®ers used as such or
after further chemical modi®cation. Estimates of
annual world production vary between 0.25 and 0.50
million tonnes. They are produced mainly by alkali-
catalysed glycerolysis of oils and fats (220±260°C,
30min). The product is a mixture of monoacylglycer-
ols (45±55%), diacylglycerols (38±45%), and triacyl-
glycerols (8±12%). The monoacylglycerol content can
be increased to over 90% through short-path distilla-
tion. The high temperatures involved in this process
make it less suitable for unsaturated compounds. The
monoacylglycerol is an equilibrium mixture of the 1-
(90%) and 2- (10%) isomers.55,56
Enzymic routes to monoacylglycerols and diacylgly-
cerols hace been studied extensively. They include
interaction of glycerol with acid, alkyl ester, or
triacylglycerol or reaction of triacylglycerol with water
(partial hydrolysis), alkyl alcohol (partial alcoholysis)
or glycerol (glycerolysis). These procedures have been
reviewed.57 Under appropriate reaction conditions it is
possible to obtain products with high levels of
monoacylglycerols without the short-path distillation
required in the existing commercial method.
McNeill et al58±61 showed that high levels of
monoacylglycerols were obtained by glycerolysis of
triacylglycerols when the reaction was carried out at
appropriately low temperatures. Monoacylglycerols
have higher melting points than other glycerol esters
and may crystallise from the reaction mixture thereby
displacing the equilibrium in favour of more mono-
acylglycerol. The reaction temperature has to be a little
below the melting point of the monoacylglycerol. This
is higher for saturated acids (52±84°C for C10 to C22)
than for unsaturated acids (oleic 35°C, linoleic
12°C).55 For example, glycerolysis of beef tallow (rich
in palmitic and stearic acids) at 48±50°C gives a
product with 30% monoacylglycerols but at 38±46°Cthis is raised to 70%. Neither solvent nor emulsi®er is
required. Pseudomonas ¯uorescens lipase is an effective
catalyst but others have also been used. Better yields
Table 4. Enrichment of EPA and DHA in fish oils
EPA, DHA
(%) Lipasea Hours Product
EPA,
DHA (%)
Tuna oil 6 and 23 Rm 24 gl esters 6 and 49
Tuna acids 6 and 23 Rm 8 acids 3 and 74
Fish oil 15 and 9 Ps 24 gl esters (50)b
a Rm=Rhizomucor miehei Ps=Pseudomonas sp.b The EPA�DHA level rises to 85% when the glycerol esters are converted to
ethyl esters (Candida antarctica) and submited to urea fractionation.
1540 J Sci Food Agric 79:1535±1549 (1999)
FD Gunstone
are obtained in a two-stage process in which reaction is
started at ca42°C for 16h and continued at 5°C for up
to four days. Under these conditions tallow, palm oil
and palm stearin were converted to monoacylglycerols
in yields of 90±95%. Similar reactions have been
reported for palm oil,62 camphor tree seed oil (a lauric
oil,63), cocoa butter,63 marine oils,64 olive oil,65,66
soybean oil67 and triolein.68
Yamane et al69 adapted the procedure to give high
yields (90%) of diacylglycerols in a two-phase glycer-
olysis of hydrogenated beef tallow effected at 60°C(2h), 55°C (4h), and then 48°C for three days.
An alternative route to mono- and diacylglycerols
involves direct reaction of glycerol with free acid or
some other source of acyl group. Macrae et al70
recommended the use of potato lipid acyl hydrolase to
promote reaction (50±70°C, 6h) between glycerol and
free acid (10:0, 12:0, 14:0, 18:1, 18:2, or 18:3) to give
monoacylglycerols in yields above 95%. Berger et al71
describe the acylation of glycerol on silica (organic
solvent, room temperature, 2±5h, in the presence of
Chromobacterium viscosum, Rhizopus delemar or Rhizo-mucor miehei lipases) with a range of acids (5:0±18:0
and 18:1). 1,3-Diacylglycerols were produced in
80±90% yield and isolated with purity >98%. The
same research group72 optimised procedures to get
high yields of monoacylglycerols (60±85%) using
excess of glycerol (1:5) and of diacylglycerols
(50±75%) using excess of acid (2:1) with the lipases
from Penicillium roquefortii or Rhizopus niveus at room
temperature. Akoh et al73 prepared monoacylglycerols
from glycerol and oleic acid (or ethyl oleate) or from
glycerol and eicosapentaenoic acid in yields of 90±98%
(Penicillium sp, molecular sieve, 25±37°C, one to four
days). Dicaprin was obtained from glycerol and ethyl
caprate in a solvent-free system in yields up to 88%
(Rhizopus arrhizus, 15±50°C).74 Edmundo et al75 pre-
pared mono-olein (71% conversion, 100% pure after
passage through a silica column) from glycerol on
silica, oleic acid, and Mucor miehei lipase in 2-methyl-
2-butanol/hexane (10:90) at 40°C.
8 TRIACYLGLYCEROLSThere is a growing demand for compounds which are
described as structured lipids. These are triacylglycer-
ols containing speci®c fatty acids in designated
positions. Laboratory processes for synthesising in-
dividual triacylglycerols are well developed76 but these
are not appropriate to produce these materials in the
volume required for nutritional purposes even in a
research exercise. Nor is it usually necessary to
produce compounds which are entirely a single species
± concentrates will usually suf®ce. Using an appro-
priate lipase as catalyst it is possible to prepare such
materials by acylation of glycerol or by deacylation/
reacylation of existing triacylglycerol mixtures. The
most important of these contain a medium-chain acid
and/or a long-chain polyunsaturated fatty acid.
Enzymic acylation of glycerol with free acid or alkyl
ester proceeds readily but the product may be mainly a
monoacylglycerol/diacylglycerol mixture rather than
triacylglycerol. This is caused by the dif®culty in
acylating the sn-2 hydroxyl group by direct acylation or
(more likely) through acyl migration.
Glycerol → 1(3)-MAG → 1,3-DAG → TAG
;1,2-DAG → TAG
(all these processes must be considered reversible)
An example is given in Table 5 for reaction between
glycerol and n-3 PUFA.77
This dif®culty does not exist when the starting point
for triacylglycerol synthesis is 2-monoacylglycerol
though care must be taken that reaction is con®ned
to acylation and that both acyl-migration and trans-
esteri®cation are kept to a minimum. This is illustrated
in the synthesis of caprucin from 2-monoerucin and
caprylic acid (Geotrichum candidum, 50°C, 75%
yield78) and in the two-step synthesis of triacylglycer-
ols described by Soumanou et al79,80 (Section 8(i)).
(i) Medium chain triacylglycerolsMedium-(M) and long-chain (L) acyl groups are
metabolised differently and since each type shows
valuable nutritional properties there is an interest in
preparing triacylglycerols with both types of acyl chain,
particularly those represented as MLM where the long
acyl chain occupies the glycerol sn-2 position. The
preparation of such compounds has been studied
intensively by Akoh and his research group. Using
Rhizopus miehei and Candida antarctica, they examined
the acidolysis and interesteri®cation of triolein, tri-
linolein, and tristearin with acids (C4, C6, and C10),
ethyl esters (C8), and triacylglycerols (C6 and C8) in
organic solvent and showed that in all cases the
resulting triacylglycerols were mainly mixtures of the
type L2M and LM2. With R miehei exchange was
con®ned almost entirely to the sn-1 and 3 positions but
with C antarctica reaction also occurred at the sn-2
position.81±87 Lee and Akoh88 studied the solvent-free
acidolysis of groundnut oil and caprylic acid in a batch
reactor on a 500g scale. The highest incorporation of
caprylic acid (c 30%) was achieved with a 1:2 molar
ratio at 50°C for 72h with Rhizomucor miehei. When
tricaprylin is submitted to acidolysis with n-3 PUFA
(Candida antarctica, 55°C, hexane) followed by short-
path distillation, structured lipids are obtained con-
taining caprylic acid (47%), eicosapentaenoic acid
(23%), and docosahexaenoic acid (22%) present in all
three of the glycerol positions.89 Capric acid and
Table 5. Composition of reaction product from glycerol andn-3 PUFA
MAG DAG TAG FFA (%)
Pseudomonas sp 24 41 18 17
Mucor miehei 34 41 13 12
J Sci Food Agric 79:1535±1549 (1999) 1541
Enzymes as biocatalysts in the modi®cation of lipids
eicosapentaenoic acid were both incorporated into
borage seed oil (already containing g-linolenic acid) in
hexane solution using the enzymes from Rhizomucormiehei and Candida antarctica as biocatalysts. Signi®-
cant proportions of both acids are incorporated but
they are found in the sn-2 position only when Candidaantarctica lipase is employed. (Table 6, Ref 90).
Kwon et al91 examined the enzymic reaction of
glycerol and capric acid (iso-octane, 25°C, up to 25h).
Non-selective lipases gave less satisfactory results than
1,3-selective lipases but not all of this latter type gave
the same results. Better results were obtained with
Rhizomucor miehei, Pseudomonas aeruginosa or Chromo-bacterium viscosum lipase) than with Aspergillus niger,Rhizopus javanicus or Rhizopus delemar lipase. The
major products were triacylglycerols but monoacylgly-
cerols and diacylglycerols were also present in the
reaction mixture. A range of PUFA were converted to
glycerol esters with lipases from Chromobacteriumviscosum (26±38% TAG) and Candida cylindracea(18±33% TAG) and more successfully with those
from Rhizomucor miehei and Candida cylindracea at
40±60°C (80±90%).92 Kosugi and Azuma93 reported
that immobilised lipase from Candida antarcticapromoted acylation of glycerol with arachidonic acid,
eicosapentaenoic acid, or docosahexaenoic acid or
their ethyl esters at 60°C. After 24h the product was
mainly TAG (80±90%) and was easily puri®ed by
passage through an alumina column. See also Refs 42
and 43.
Acylation of glycerol on silica gel with capric acid in
iso-octane at 40±60°C has been examined with a range
of lipases. Tricaprin was the major product with some
lipases (Pseudomonas aeruginosa, Rhizomucor miehei,and Chromobacterium viscosum) and dicaprin with
other lipases (Candida rugosa and Rhizopus delemar).94
With Rhizomucor miehei (60°, 5h) Ghosh et al95 raised
the levels of caprylic and capric acids in coconut oil or
coconut olein by interesteri®cation with the methyl
esters of these short-chain acids from ca12 to 20±25%.
Gioielli et al96 have described the modi®cation of
babassu oil (a lauric oil) by acidolysis with palmitic
acid (raised from 10 to 22%, Rhizomucor miehei, 65°C,
4h).
A group working in Stuttgart79,80,97 have shown that
MLM triacylglycerols are best made in a two-step
process. Triacylglycerols (triolein, trilinolein, or
groundnut oil) are ®rst subject to ethanolysis (Rhizo-pus delemar, 40°C, 30h, MeOBut-hexane solution) to
give a 2-monoacylglycerol. This is puri®ed by crystal-
lisation from hexane (72% yield of recovered product)
and then acylated (Rhizomucor miehei, caprylic acid,
hexane, molecular sieve, 38°C) to give a ®nal product
containing caprylic acid (94%) in the sn-1 and 3
positions and unsaturated C18 acid (98%) in the sn-2
position.
Xu et al98,99 have described studies to optimise the
acidolysis reaction (®sh oil and capric acid or medium-
chain triglycerides and sun¯ower acids) in a one-
kilogram reactor with emphasis on the minimisation of
acyl migration.
(ii) Triacylglycerols enriched in EPA and/or DHAStructured lipids containing eicosapentaenoic acid
and/or docosahexaenoic acid can be made in several
ways: (i) by reaction of glycerol with polyunsaturated
fatty acids or their ethyl esters, (ii) by enrichment of
acids already present in ®sh oils by appropriate
deacylation and reacylation procedures, and (iii) by
reaction of vegetable or other oils with PUFA con-
centrates as free acids, ethyl esters or glycerol esters.
Acids/esters enriched in eicosapentaenoic acid and/or
docosahexaenoic acid are often obtained from ®sh oils
by urea fractionation of the acids or alkyl esters.
Glycerol and a concentrate of polyunsaturated fatty
acids (24% EPA and 53% DHA) reacted in organic
solvent under the in¯uence of Chromobacterium visco-sum lipase to give a mixture of monoacylglycerols
(14%, with 27% EPA and 50% DHA), diacylglycerols
(43%, with 25% EPA and 50% DHA), and triacylgly-
cerols (37%, with 21% EPA and 50% DHA).100
Cerdan et al101 used the lipase from Candida antarcticato convert glycerol and a PUFA concentrate (26%
EPA and 48% DHA) from cod liver oil into a product
which was 85% triacylglycerol with 26% EPA and
45% DHA (60°C, 96h).
Several papers describe the concentration of eico-
sapentaenoic acid and docosahexaenoic acid in ®sh
oils by partial hydrolysis with a lipase that discrimi-
nates against DHA (and EPA). These acids concen-
trate in the residual glycerol esters which are generally
mixtures of di- and triacylglycerols that can be acylated
to triacylglycerols. Reference has already been made to
work by a number of researchers41±47 on the concen-
tration of docosahexaenoic acid (Section 5). Tanaka etal102 converted tuna oil (25% DHA) by partial
hydrolysis (Candida antarctica) followed by acylation
with PUFA from tuna oil (Chromobacterium viscosum,
50°) to triacylglycerols with 39±46% of DHA. Maehr
et al103 used lipase from Pseudomonas sp at ambient
temperature to raise the level of n-3 PUFA from 29±
34% to 50% with 23±50% recovery or to over 70%
with 14±22% recovery.
Yamane et al104,105 used the lipase from Mucormiehei or Candida cylindracea to raise the levels of EPA
and DHA in cod liver oil (9 and 13%) to 22 and 34%
when the reaction was conducted at ÿ10°C for 20h
followed by ÿ20°C for 40h.
Sridhar et al106 incorporated EPA (9%) and DHA
Table 6. Distribution of selected acids in borage oil afteracidolysisa
10:0 18:2 g-18:3 20:5
Borage ± 39 (47) 20 (20) ±
Modi®ed Rm 26 (±) 21 (49) 17 (22) 10 (±)
Modi®ed Ca 16 (8) 27 (50) 17 (15) 9 (5)
a Levels of 10:0 and 20:5 in the treated borage oil
(percentage in the sn-2 position).
1542 J Sci Food Agric 79:1535±1549 (1999)
FD Gunstone
(8%) into groundnut oil using PUFA with 35 and 20%
of these two acids and Mucor miehei lipase for 4±6h.
Akoh and his colleagues85,107,108 incorporated EPA
and/or DHA into several vegetable oils (canola,
groundnut, soybean, hydrogenated soybean and
high-oleic sun¯ower), and trilinolein, tricaprylin,
tricaprin, and trilaurin with Mucor miehei and Candidaantarctica lipases at 55°C for 24h. Lee et al85 showed
that these enzymes incorporate 35±42% of EPA into
short- and medium-chain triacylglycerols but they
behave differently (Table 7). With Mucor miehei EPA
was con®ned to the sn-1(3) position but with Candidaantarctica it occurred in all three glycerol positions.
(iii) Margarine and shorteningMargarines and shortenings made with partially
hydrogenated fats contain acids with trans unsatura-
tion which are out of favour on nutritional grounds.
The melting behaviour required of a spread can be
achieved, at some small cost in terms of increased
saturated acids, by interesteri®cation of suitable blends
of hard stock and vegetable oils and this change is
usually effected using an alkaline catalyst.109,110
Similar changes can be achieved with appropriate
lipases and several procedures have been described.
For example, a product with melting behaviour
appropriate for use as a margarine or shortening was
made by enzymic interesteri®cation of a high-melting
palm stearin (mp 58°C) with a vegetable oil (sun-
¯ower, soybean, or rice bran) in 40:60 ratio (Mucormiehei, 60°C, 5h). The product contained polyunsa-
turated fatty acids and was virtually free of transacids.111 When lard and high-oleic sun¯ower (60:40)
were interesteri®ed (Candida antarctica, 55°C, 24h)
the product was superior to that obtained by blending
alone and had solid content resembling that of a soft
margarine at a range of temperatures.112 From the
examination of a ternary mixture of canola oil,
hydrogenated canola oil, and palm stearin Cho and
deMan113 concluded that the interesteri®cation pro-
duct crystallised in the desirable b' form and could be
used to produce margarine with trans levels lower than
conventional products made from hydrogenated oils
even though trans acids were still present.
Foglia et al114 have reported changes in melting
behaviour resulting from enzymic interesteri®cation of
tallow, high-oleic sun¯ower oil, or tallow-sun¯ower
mixtures (Mucor miehei, Rhizopus delemar, or Geo-trichum candidum at 50±70°C). They conclude that
glyceride composition can be in¯uenced by choice of
enzyme under controlled conditions in contrast to
chemical reaction products that are wholly random in
nature.
Graille et al115 have studied the interesteri®cation of
palm oil and palm stearin with several other oils using a
1,3-regiospeci®c lipasae (Mucor miehei, 60°C). They
obtained products which, depending on the conditions
employed, can be used for ®rm or soft margarines.
(iv) Cocoa butter equivalentsEnzymic interesteri®cation of oils and fats to produce
cocoa butter equivalents has been reviewed by
Quinlan and Moore116 and by Rosendaal and
Macrae109 and is covered by patents on these materials
to Unilever and to Fuji Oil Co. The production of
useful products has been described by Bloomer et al117
(palm mid fraction and ethyl stearate), Chang etal118(hydrogenated cottonseed oil and olive oil), and
by Liu et al119 (palm oil and tristearin).
9 WAX ESTERS, SUGAR ESTERS, AND ESTERSOF POLYHYDRIC ALCOHOLS OTHER THANGLYCEROLLipases from Rhizopus miehei or Candida antarcticahave been used to promote acylation of medium-chain
(8:0) and long-chain (18:0) alcohols with the acyl
group provided as free acid, methyl ester, or triacyl-
glycerol. Reaction occurs at ca 60°C and high yields
(95±100%) of wax esters are obtained in 30±
120min.120±123 Krmelj et al124 examined the lipase-
catalysed synthesis of oleyl oleate in pressurised and
supercooled solvents using Rhizomucor miehei lipase.
In butane 87% conversion was obtained (20°C, 20
bar, 5h) while carbon dioxide gave 86% conversion in
one hour (50°C, 100 bar).
Hayes and Kleiman125 described the Lypozyme-
catalysed reaction between diols and either lesquerella
oil or lesquerolic acid to give mono- and diesters which
have potential application as cosmetics and as lubri-
cants. The secondary hydroxyl group in lesquerolic
acid is not involved in the process.
Sugar estersIt is possible to prepare lipid-like molecules by
replacing glycerol with mono- or disaccharides. The
acylated sugars have interesting nutritional properties
and can also be used as surfactants.126 The best known
is olestra, a sucrose derivative with six to eight
esteri®ed groups obtained from sucrose and common
vegetable oils (soybean, corn, cottonseed, sun¯ower,
rapeseed). It is prepared by reaction of high-grade
sucrose with high-grade methyl esters in the presence
of sodium or potassium soaps to improve solubility
and an alkali carbonate as catalyst. It can be used as a
frying oil and can replace fats in products such as ice
cream, margarine, cheese and baked goods. The
material is non-toxic, non-carcinogenic, and is so
poorly absorbed as to have zero calori®c value.
Permission has been granted for limited use in the
Table 7. Incorporation of EPA into short- and medium-chaintriacylglycerolsa
Mucor miehei Candida antarctica
Tricaprylin 34 (±) 38 (35)
Tricaprin 32 (±) 42 (39)
Trilaurin 36 (±) 41 (36)
a Values of EPA incorporated into the glycerol ester (EPA in
the sn-2 position).
J Sci Food Agric 79:1535±1549 (1999) 1543
Enzymes as biocatalysts in the modi®cation of lipids
USA and on balance it has been well received but it is
not yet licensed for use elsewhere.
Partly acylated sugars with a balance of hydrophobic
and hydrophilic groups show interesting surfactant
properties. Attempts to produce such molecules by
standard chemical syntheses are likely to be tedious
and instead, attempts have been made to exploit the
speci®city of lipase. Mutua et al127 described the
acylation of methyl glucoside and related alky glyco-
sides with methyl oleate (Candida sp, 55°C, 24±48h,
benzene-pyridine) with 60±100% incorporation of the
acyl group. The same research group acylated glucose
penta-acetate with methyl oleate or high-oleic sun-
¯ower oil as the acylating agent in the presence of
lipases from Candida antarctica and Candida rugosa.128
In a more controlled procedure Sarney et al129
achieved the regioselective synthesis of sucrose
monoesters (6'O-acyl- and 6-O-acyl) by lipase-cata-
lysed acylation (Candida antarctica and Rhizomucormiehei, 48h, 75°C, range of organic solvents) of
appropriate sucrose acetals in which secondary hydro-
xyl groups are blocked leaving the primary hydroxyl
groups free for acylation.
The Stuttgart group130 has shown that drawbacks
like low solubility, low reaction rate, and low
productivity associated with the enzymic biotransfor-
mations of polar sugars in dilute organic solvents can
be easily overcome even when carrying out these
reactions in a system with solid substrates and
products. The correct choice and concentration of
the organic solvent serving as adjuvant is most
important to obtain high yields of sugar fatty acid
esters. Typical reaction conditions leading to good
results (up to 100% acylation of primary hydroxyl
group) require acetone, Candida antarctica, 12:0±18:0
fatty acids, 48h. A clear advantage of this reaction
system is the use of readily available starting materials
and no activated acyl donors or modi®ed sugars in
order to achieve high conversions in acceptable
reaction times.
Other estersAcylated derivatives of propylene glycol (1,2-propane
diol) are mixtures of mono-(mainly 1-) and di-esters
and are used as food-grade surfactants. With propy-
lene glycol, Pseudomonas lipase, and organic solvent
(hexane, toluene) at 30°C the product was mainly the
mono-ester obtained in yields of 29±37% (with acids),
65±86% (with triacylglycerols), and 80±90% (with
anhydrides) depending on the nature of the C12±C18
acyl group.131 The same group have prepared propy-
lene glycol esters containing EPA and DHA.132
Trimethylolpropane (TMP) esters, used as bio-
degradable lubricants, are normally made through
high-temperature acylation. They can, however, be
obtained through alcoholysis of rapeseed methyl esters
with TMP using lipase from Candida rugosa.133
The glycerol ether 1-O-hexadecylglycerol can be
acylated by C14±C18 fatty acids in the presence of
Candida cylindracea or Pseudomonas sp lipases (di-
chloromethane, three days at 33°C or for a shorter
time at 55°C) to give a 75% yield of a 7:1 mixture of
the 3- and 2-acyl ethers.134
10 AmidesLipases from Rhizopus miehei and Candida antarcticahave been used to promote acylation of amino
compounds to give amides using triacylglycerols or
methyl esters as the source of the acyl group. Examples
include the N-acylation of lysine,135 butylamine and
other amines,136 and b-alanine.137
11 CHEMICAL REACTIONS OF THE ALKYL CHAINGardner138 reviewed the role of lipoxygenase as a
versatile biocatalyst and Gargouri and Legoy139
illustrated its use in the preparation of (�)-coriolic
acid (13S-OH 9c11t-18:2) from trilinolein or from
sun¯ower oil used as a rich source of linoleic acid. The
triacylglycerols were ®rst hydrolysed by lipase from
Pseudomonas sp to the free acids which were then
oxidised by lipoxygenase to the 13-hydroperoxy acid
and reduced chemically (NaBH4-EtOH) to the hydro-
xy acid (purity >95%, yield 17±78% depending on
concentration of triacylglycerol). Reaction occurs at
pH 9 and 28°C in 150±200min in a two-phase system.
Hydrolysis occurs at the interface and oxidation in the
aqueous phase.
ÐCH=CHCH2CH=CHÐ →
CH(OOH)CH=CHCH=CHÐ →
CH(OH)CH=CHCH=CHÐ
By reaction with hydrogen peroxide in toluene in the
presence of Candida antarctica lipase triacylglycerols or
methyl esters are converted to peroxy acids which then
effect epoxidation of unsaturated centres.140 With
rape, sun¯ower, and linseed oils epoxidation is about
90% complete. Frykman and Isbell141 applied a
similar procedure to meadowfoam fatty acids, rich in
D5 acids (30% hydrogen peroxide in toluene or
dichloromethane and Candida antarctica lipase) and
subsequently converted the epoxy acids to hydroxy
lactones and estolides by chemical processes.
Bacillus lentus NRRL B-14864 converts 12-hydro-
xystearic acid (from ricinoleic acid) to a C12 tetra-
hydrofuran acid, via chain-shortening (b-oxidation) to
6-hydroxydodecanoic acid. This intermediate under-
goes cyclisation at the 2t or 3-hydroxy stage during the
next oxidation cycle.142
In the presence of appropriate enzyme sources, oleic
acid and other appropriate acids are converted to a
range of oxidation products which are not easily
produced with chemical reagents. Oleic acid is
1544 J Sci Food Agric 79:1535±1549 (1999)
FD Gunstone
converted to 15-, 16-, and 17-hydroxyoleic acids (5±
11%) with Bacillus pumilis in three to ®ve days at
32°C143 and to 7R,10R-dihydroxy 8t-18:1 with
bacterial strain PR3.144 With Pseudomonas aeruginosaPR3 the yield of dihydroxy acid was 80%145 and this
compound can be reduced with hydrazine to 7R,10R-
dihydroxystearic acid. Linoleic acid did not react in
this system but ricinoleic acid gave 7,10,12-trihydroxy
8t-18:1 (35% yield) by an analogous process.
Flavobacterium sp DS5 hydratase converts oleic acid
to a mixture of 10-hydroxy and 10-oxo-stearic acid.
Other D 9c acids (9c-14:1, 9c-16:1, linoleic, and a- and
g-linolenic acids) react similarly to give appropriate
10-hydroxy and oxo acids but there is no reaction with
elaidic (9t-18:1), erucic (13c-22:1), arachidonic (all-cis5,8,11,14±20:4), or petroselinic (6c-18:1) acids, all of
which lack the D 9 cis double bond.146
ÐCH=CHÐ → ÐCHOHCH2Ð → ÐCOCH2Ð
The microbial culture Clavibacter sp ALA2 converts
linoleic acid to 12,13,17-trihydroxy 9c-18:1 as major
product (25%) accompanied by minor products (each
3±4%) which have been identi®ed as the tetrahydro-
furan derivatives: 12-hydroxy- and 7,12-dihydroxy-
13,16-epoxy 9c-18:1.147,148
12 REVIEWSSeveral aspects of this topic have been reviewed pre-
viously. Malcata149 described the use of immobilised
enzymes in the modi®cation of oils and fats by
hydrolysis, ester synthesis, and interesteri®cation.
Immobilisation procedures, reactor con®guration,
and process considerations are also discussed. Born-
scheuer57 has described the lipase-catalysed synthesis
of monoacylglycerols, and the ability of lipases to
convert hydroxy acids to lactones and estolides is
considered by Hayes.150 Gandhi151 has produced a
useful review on lipase applications covering both
hydrolysis and ester synthesis. Other reviews devoted
to lipases are by Quinlan and Moore116 and by
Rosendaal and Macrae.109
Uhlig152 in his book ªIndustrial Enzymes and theirApplicationsº includes a section on lipases, including
animal lipases, microbial lipases, and phospholipases.
The most recent review of this topic is a chapter on
enzymic processes by McNeill153 who is also an author
of several signi®cant papers cited in this review. After a
discussion of the enzymes themselves, his review is
focused on potential products (triacylglycerols, di-
acylglycerols, monoacylglycerols, fatty acids, phos-
pholipids, sugar esters, and waxes).
In his review of the role of lipoxygenase as a versatile
biocatalyst Gardner138 includes details of the chemical
and enzymic reactions of hydroperoxides, the latter
with allene oxide synthase and hydroperoxide lyase.
Over 100 compounds have been identi®ed from
linoleic acid and related compounds are obtained
from other PUFA.
13 CONCLUSIONInterest in the use of lipases and other enzymes to
promote reactions, some of which are not easily
effected by non-enzymic processes, has grown con-
siderably in the last 10 years in both academic and
industrial laboratories. This is re¯ected in a rapidly
growing number of publications. At the present time
interest is focussed on the use of these systems to
concentrate and isolate polyunsaturated fatty acids
required for dietary studies in animals and in humans
and on the production of structured lipids. The latter
are triacylglycerols (and phospholipids) with speci®c
fatty acids or fatty acid classes esteri®ed with glycerol
in speci®c positions generally on the basis of their
nutrirional properties. One can expect further devel-
opments in the next ten years and the arrival of more
products in the market place.
REFERENCES1 Gunstone FD, Movements towards tailor-made fats. Prog Lipid
Res 37:277±305 (1998).
2 Hilditch TP, The Industrial Chemistry of Fats and Waxes, Balliere,
Tindall and Cox, London, pp 235±237 (1927).
3 Holmquist M, Insights into the molecular basis for fatty acyl
speci®cities of lipases from Geotrichum candidum and Candida
rugosa. Chem Phys Lipids 93:57±65 (1998).
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