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using lipase B from Candida antarctica. Eur. J. Lipid Sci. Technol. 106, 513–517.

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CHAPTER 20

USE OF LIPASES IN THE SYNTHESIS OF STRUCTURED

LIPIDS IN SUPERCRITICAL CARBON DIOXIDE

JOSÉ DA CRUZ FRANCISCO

, SIMON P. GOUGH

AND ESTERA S. DEY

1∗

Department of Pure and Applied Biochemistry and

Department of Biochemistry, Lund University, Lund, Sweden

∗

estera.dey@tbiochem.lth.se

1. INTRODUCTION

Interesterification is one of the techniques used to obtain oils or fats with

desirable physicochemical characteristics. Additionally, interesterification can

produce oils/fats with useful nutritional and health attributes such as the struc-

tured fats. The interesterification can be performed both chemically and enzymat-

icaly. However, enzymatic interesterification of oils and fats using immobilized

lipases has attracted especial attention because of the lower operation temperature

required and the yield of a more defined product.

In recent years a more environmentally accepted reaction medium for such

reactions has been sought. This focused attention on enzymatic interesterification

in supercritical fluids, particularly supercritical carbon dioxide scCO

, to replace

organic solvents. scCO

as a reaction medium principally offers the same advan-

tages for lipase catalysis as organic solvents. Hydrophobic lipid substrates are

soluble, immobilized lipase is easily recovered and the reversal of hydrolysis

favours the synthesis (Martins et al., 1994; Gunnlaugsdottir and Sivik, 1997).

However scCO

provides even more advantages, see section 6.1.

This chapter discusses the use of lipase in scCO

for the transesterification

reactions and explores the advantages, drawbacks and difficulties encountered in the

attempt to produce structure oils/fats.

2. STRUCTURED LIPIDS

In this chapter, the terms lipid or structured lipids will be restricted to triacylglycerols

which include oils and/or fats commonly used for food purposes. Structured lipids

(SL) are defined as triacylglycerols containing medium (MCFA) or short-chain fatty

341

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 341–354.

342 JOSÉ DA CRUZ FRANCISCO ET AL.

acids (SCFA) and long-chain essential/functional fatty acids produced by chemical

modification orenzymatic synthesis. Structured lipids thus provide botha swift energy

source as well as essential fatty acids. The idea of using structured lipids for nutritional

applications arose from successful clinical applications of medium-chain triacylglyc-

erols. Further research showed that the inclusion of both medium chain fatty acids

and essential fatty acids in the same triacylglycerol improved the nutritional effec-

tiveness of SL (Xuebing et al., 2002). Before absorption, triacylglycerols containing

longchain fatty acids (LCFA)(>12 carbon atoms) must be hydrolyzedin the intestinal

lumen. Subsequently, they must be re-synthesized in the enterocytes and then used

to form lipoprotein particles known as chylomicrons. From the chylomicrons, long

chain fatty acids (LCFAs) are released by serum lipases and incorporated in the

tissues, stored in the adipocytes or burnt in the mitochondria. However, before uptake

across the mitochondria membrane, LCFA must be converted to acylcarnitine by the

acyltransferase.IncontrasttriacylglycerolscontainingMCFAcan directlybeabsorbed

without hydrolysis and preferentially transported through the portal venous system

to the liver. Thus, MCFA are readily burnt via mitochondrial -oxidation whereas

mostLCFAare incorporated into triacylglycerolsin the hepatocyte. So,dietary MCFA

intervention bypasses lipid deposition into adipocytes, the lipase-regulated metabolic

pathway of LCFA. This can reduce the tendency to obesity (Mu et al., 2005). When

22% n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) are also included

in the diet endogenous oxidation of LCFA has also been demonstrated (Beermann

et al., 2003). Compared to LCFA, fats containing some MCFA, have been shown to

reduce bodyweight in rats (Shinohara et al., 2005). MCFAsare metabolized as rapidly

as glucose in the body. Because they are not readily re-esterified into triacylglyc-

erols, there is little tendency to deposit as stored fat. MCFAs are therefore useful in

the control of obesity. However, they can potentially raise serum cholesterol levels.

MCFAs in a structured lipid substituted to glycerol at position sn-1, and sn-3 are better

and more useful. Such lipid construction combines their inherent mobility, solubility,

and easy metabolism. With beneficial polyunsaturated fatty acids located in position

sn-2 it is possible to achieve good nutritional properties (Osborn and Akoh, 2002).

The composition of the triacylglycerols in each fat or oil is specific to its origin.

The physical properties of the various fats/oils are directly related to the structure

and distribution of the fatty acids in the triacylglycerol backbone (Parodi, 1982;

Macrae, 1985; Christie, 1986; Willis et al., 1998). Naturally occurring fats or oils

are mixtures of triacylglycerols. In such mixtures triacylglycerols with an ideal

distribution of the fatty acids in the glycerol backbone corresponding to a structured

lipid are not common and therefore they must be obtained artificially.

3. SYNTHESIS OF STRUCTURED LIPIDS VIA LIPASE

Structured lipids can be produced by chemical or enzymatic interesterification.

Whether chemically or enzymaticaly, during interesterification there is a

redistribution of acyl groups of the triacylglycerols participating in the reaction to

achieve the desired structures (Willis and Marangoni, 1998).

USE OF LIPASES IN SYNTHESIS OF STRUCTURED LIPIDS 343

The first studies on interesterification were published early in the 18th century.

At the present interesterification plays an important role in the production of

low caloric fats replacers (Smith et al., 1994; Rousseau and Marangoni, 1998a).

Interesterification include; reactions between a triacylglycerol and a fatty acid

(acidolysis), between an alcohol and a triacylglycerol (alcoholysis), glycerolysis

(when glycerol acts as the alcohol) and between two triacylglycerols (trans-

esterification) (Feuge, 1962; Sonntag, 1982; Malcata et al., 1990; Willis and

Marangoni, 1998).

Transesterification is the most common type of interesterification in food

applications. Chemical interesterification has been used for many years but research

has presently directed to replace it by enzymatic interesterification. There are two

reasons for this. Chemical interesterification requires severe reaction conditions

and produces positional randomization of the acyl groups in the triacylglycerols

obtained. Conversely, many factors recommend enzymatic interesterification.

Enzymes work well under mild conditions, specifically produce well defined

products and produce less waste. Reusable immobilized enzymes lead to more

economical processes (Goh et al., 1993).

4. MOLECULAR STRUCTURE AND MECHANISM OF LIPASES

Lipases can be divided into two general structural classes: those which exist in an

inactive form, where the active site is covered by a lid and those where the active

site is always exposed to solvent. Examples of the commercially available classes

of such enzymes are presented in Table 1. The lipozyme of Mucor miehei RM-IM

is one of the lipases with movable lid which have been used in synthesis purposes

in supercritical carbon dioxide. This lipase from Novozymes, has been immobilized

on a weak-anion-exchange resin (IM) (Boel et al., 1988). It can rearrange the fatty

acids in the sn-1 and sn-3 positions of triglycerides. Only the sn-2 positions are

preserved. It belongs to the alpha/beta hydrolase fold enzyme family with an active

site triad of serine aspartate and histidine similar to serine proteases (Holmquist,

2000). Kinetic experiments suggested that lipase activation involved the formation

of a local chamber on phase contact area where the hydrolysis occurs. Three regions

were suggested in the active centre of lipolytic enzymes: 1) a region responsible

for the recognition” of substrate phase surface; 2) a binding region, participating in

hydrophobic interaction with a single substrate molecule, located in the insoluble

phase; and 3) catalytical region (Chapus and Semeriva, 1976).

The crystal structure of Mucor miehei lipase (Brzozowski et al., 1991) shows that

the serine esterase inhibitor p-nitrophenyl diethyl phosphate has reacted with the

S144 to give a serine bound diethyl phosphate. This identifies S144 as the active site

serine. The C-terminal part of the un-liganded lipase structure can be superimposed

on the liganded structure (Fig. 1). However the N-terminal short amphiphilic helix

(residues 85 to 91) acts as a “closed hydrophobic lid” in the unliganded structure.

The hydrophobic residues (white) of the amphiphilic helix are internalised and the

polar residues (black) solvent exposed (Fig. 1 top left). The hydrophobic residues

344 JOSÉ DA CRUZ FRANCISCO ET AL.

Table 1. Commercially available lipases used in synthesis of structure lipids

Commercial name Lipase Lipase class

Novozym

435 Immobilized Candida antarctica

lipase B

Permanently open active site

Lipozyme RM IM

Mucor miehei immobilized Active site covered by

movable lid

Lipozyme

TM IM Thermomyces lanuginosus on

porous silica

Active site covered by

movable lid

Lipex

100L Thermomyces lanuginosus Mutant

with better lipid susface

adsorption (Snab, 2004)

Active site covered by

movable lid Stable to

detergents

Palatase

20000 L, Mucor miehei(Barron et al., 2004) Active site covered by

movable lid

Novozym CALB L

Candida antarctica lipase B Permanently Open active site

Lipozyme

TL100 L Thermomyces lanuginosus Active site covered by

movable lid

lie above the active site histidine and serine. This lid is inverted in the inhibitor

bound structure. The helix extended by the hydrophobic residue 92, moves away

from the catalytic triad and turns outward to face the solvent to give the “open”

structure (Fig. 1 top right). The active site residues can be viewed from above as

in Figs. 2 and 3, surrounded by the solvent accessible >30%) residues. It can be

clearly seen that non-polar residues are clustered on the top compared to the bottom

of these pictures. In the orientation of these pictures the active site is partially

buried on top by the internalised (<30% accessible) hydrophobic residues I204 and

V205 whereas on the bottom, solvent access to active site histidine and aspartate is

unrestricted (not shown). The main difference in the liganded structure (Fig. 1 top

right), compared to the unliganded (Fig. 1 top left), is the presence of the non-polar

solvent-exposed patch formed by hydrophobic residues in the amphiphilic helix.

This patch is considered to facilitate contact with lipid droplets allowing product

release and substrate binding. In the liganded structure the strongly conserved W88

of the amphilic helix, although exposed to solvent, remains close to the hydrophobic

ethyl groups of the inhibitor and presumably interacts hydrophobically with the non-

polar. A completely analogous behaviour of the corresponding amphiphilic helix

is seen in the structure of the active site S144A mutant Thermomyces lanuginosa

lipase. In this case the open lid structure is seen with bound oleic acid and the closed

lid with the unliganded enzyme (Brzozowski et al., 2000). These fungal lipases

are assigned to the class Lipase 3. Conservation of the catalytic triad and sequence

similarity of lipases from these fungi and from organisms including viruses, bacteria,

plants and other eukaryotes including plasmodium and homo suggests that these all

have a similar structure and mechanism.

A simple two state model of the lipase suggested that closed state might be

more stable in aqueous media rendering the active site inaccessible to water soluble

substrates. However the lid of the Humicola lanuginosa (now called Thermomices

USE OF LIPASES IN SYNTHESIS OF STRUCTURED LIPIDS 345

Figure 1. Superinposed of unliganded and liganded (bound to p-nitrophenyl diethyl phosphate) struc-

tures of Mucor miehei lipase. Top left: the hydrophobic residues (white) of the amphiphilic helix are

internalised and the polar residues (black) solvent exposed. Top right: the helix moves away from the

catalytic triad and turns outward to face the solvent

langinosus) lipase is disordered irrespective of the media’s ionic strength. Rhizopus

delemar lipase crystallized in detergent exists in an asymmetrica dimeric form and

both open and closed lid forms are seen. These new results call into question the

Figure 2. Liganded (bound to p-nitrophenyl diethyl phosphate) open structure of Mucor miehei lipase.

Hydrophobic and hydrophilic residues are represented in white and black, respectively

346 JOSÉ DA CRUZ FRANCISCO ET AL.

Figure 3. Unliganded closed structure of Mucor miehei lipase. Hydrophobic and hydrophilic residues

are represented in white and black, respectively

simplicity of the enzyme theory of interfacial activation of Penicillium camemberti

and Rhizopus oryzae lipases (Derewenda et al., 1994a, b).

The structure of a mutant M211S/R215L with changed substrate specificity shows

an unexpected dimerization and domain sapping involving the N-terminal region.

When a tailored substrate-like irreversible inhibitor 1-hexadecanesulfonyl chloride

binds, a cis-trans isomerization of the F37-P38 peptide bond negates this dimer-

ization. Different structural rearrangements of an open to a closed form are a general

property of other lipases (DeSimone et al., 2004a). Kinetic analysis suggests that

short chain p-nitrophenyl-hexanoate substrates are metabolised by a different kinetic

mode than long chain ones e. g. p-nitrophenyl dodecanoate. This was confirmed

by different binding modes in the structures(DeSimone et al., 2004b). The main

conclusion is that, although it would be expected that the active form of the movable

lid lipase be more stable in solvent of lower ionic strength, organic solvents or

scCO

, this remains to be conclusively demonstrated.

5. REACTIONS INVOLVING LIPASE IN ScCO

Since Randolph et al. (Randolph et al., 1985) and Nakamura et al., (Nakamura et al.,

1985) established the stability and activity of enzymes in supercritical fluids, several

lipase reactions in scCO

have been described. Examples include esterification of

free fatty acids and primary alcohols (Steytler et al., 1991; Marty et al., 1992b; Yu

et al., 1992), alcoholysis of propylacetate with geraniol (Chulalaksananukul et al.,

1993) and cod liver oil and ethanol (Gunnlaugsdottir and Sivik, 1995) as well as the

acidolysis of triolein and stearic acid using a 1,3-specific regiospecific lipase (Chi

et al., 1988; Nakamura, 1994). Transesterification of triolein with ethylbehemate by

immobilized lipase (Yoon et al., 1996) as well as the transesterification of different

triglycerides (milk fat and canola oil) (Yu et al., 1992) were also reported.

USE OF LIPASES IN SYNTHESIS OF STRUCTURED LIPIDS 347

6. FACTORS INFLUENCING LIPASE ACTIVITY

AND SYNTHESIS IN ScCO

The pH, temperature, water content and activity, enzyme purity and presence of

other proteins, substrate composition and steric hindrance, surface active agents

and product accumulation are factors that can influence the lipase activity during

interesterification. These factors have been exhaustively discussed in an elegant

review by Willis and Marangoni (Willis and Marangoni, 1998). However, this

review does not include processes where supercritical fluid is the reaction medium.

Many studies involving interesterification in scCO

have been performed and the

factors affecting the reaction in organic media have been investigated.

A fluid is considered to be supercritical when its temperature and pressure

are above the critical point; the maxima pressure and temperature combination

in which the equilibrium gas-liquid is possible. Because supercritical fluids are

good solvents, they have received great attention as possible new solvents for

enzymatic reactions since the mid-1980’s (Nakamura, 1990; Aaltonen, 1991; Marty

et al., 1992a). This is because supercritical fluids have physical chemical properties

between those of liquid and gas. The high diffusivities, lower viscosities and lower

surface tension relative to liquids, accelerate the reaction rates. Low viscosity

and high diffusivity of the reaction medium, for instance, speed up reactions

controlled by mass transfer (Stahl et al., 1988); e.g. lipase in scCO

is about 8–

9 fold more efficient than in hexane has been reported. In scCO

Mucor miehei

immobilized lipase esterified mystiric acid and ethanol with an average efficiency

of 38–68% compared to 4–9% in hexane. This large increase in efficiency was

attributed to the superior rate of diffusion of mysteric acid in scCO

where its

diffusion coefficient is hundred times higher than in hexane (Bernard et al.,

1995). Because of its relatively low critical point (31



C; 7.3MPa), scCO

has

been widely explored as reaction medium. A low critical temperature allows

enzymatic reactions to occur even at physiological temperatures; a good feature for

biocatalysis. Furthermore, the readily available CO

is non-volatile, non-flammable,

and non-toxic. scCO

has excellent properties for lipase reactions (Nakamura, 1990;

Aaltonen and Rantakyla, 1991; Russell et al., 1991) and the possibility of changing

the physical state by simple manipulation of the pressure and temperature facilitates

product recovery. This makes scCO

specially attractive for food and pharmaceu-

tical industrial processes (Marty et al., 1994) and it is environmentally accepted

(Francisco et al., 2003a).

6.1. Enzyme Stability in ScCO

Enzymes are proteins. Proteins are practically insoluble in supercritical CO

However, enzyme stability must be considered since in esterification and inter-

esterification reactions, water, heat and additionally when using supercritical CO

pressure are involved.

In scCO

pressure is not a threat to protein configuration since it is only

irreversibly influenced at pressure much higher (600 MPa and higher) than those

348 JOSÉ DA CRUZ FRANCISCO ET AL.

used (Bridgman, 1914; Aoki et al., 1968; Murphy, 1978; Chryssomallis et al.,

1981; Kornblatt et al., 1982; Kornblatt et al., 1986). When protein denaturation

was observed at lower pressures and temperatures it has been attributed to water

content and heat rather than pressure (Weder, 1990).

After six days of incubation at pressure from 13 to 18MPa at 40



C, Marty and

co-workers (Marty et al., 1990) observed only a 10% decay of the enzyme activity.

At 9.6MPa and 70



C, Lipozyme RM IM retained complete activity for more than

26 hours (Bister, 2004). Loss of enzyme activity during the depressurization step

has been reported (Kasche et al., 1988; Marty et al., 1992b), however this effect

was only observed in hydrated scCO

The good stability of the enzyme makes this technique attractive in the pharma-

ceutical field where mild operation temperatures and solvent safety is crucial.

6.2. Water Activity

Water influences the hydration of the enzyme and consequently the conformation and

optimal activity (Rupley et al., 1983; Zaks et al., 1988; Klibanov, 1989; Chulalak-

sananukul et al., 1990; Hirata et al., 1990; Chulalaksananukul et al., 1992; Halling,

1994. The water content of the solid support is deemed to be the most important

aspect for the enzyme activity. In esterification, water is one of reaction products

whoseeffect inthe thermodynamicofthe reactionis predominant(Marty etal., 1992a).

Chulalaksananukul and co-workers (Chulalaksananukul et al., 1993) have

investigated the effect of addition of water and increased temperature (40 to 100



at 14MPa) on the stability of immobilized lipase from M. miehei in scCO

. The more

water added and the higher the temperature applied, the lower the residual activity

of the enzyme. For instance, by adding 30l to 100mg of enzyme, the residual

activity dropped from 70% at 40



C to 30% at 100



C; cf Fig. 2 (Chulalaksananukul

et al., 1993). The decrease of enzyme activity with the water increase was evident

for every temperature tested. From the same results it was observed that even

without water addition, the enzyme activity decreased solely as a consequence of

temperature increase. The authors also investigated the influence of the added water

on the lipase catalyzed interesterification of geraniol and propyl acetate. The initial

rate increased with increasing the amounts of water added to a maximum value at

3l and 10l of water in hexane and scCO

, respectively and then a progressive

decreasing was observed. The difference between the optimum water amounts in

both solvents was attributed to the fact that scCO

is less hydrophobic than hexane.

Quantification and comparison of the effect of water content in organic solvent

and scCO

and its relationship to pressure and temperature have been investigated

(Marty et al., 1992b).

6.3. Temperature and Pressure

The physical properties of scCO

depend greatly on the pressure and temperature.

It is conveniently possible to tune its solvent power and thus its behavior as a

USE OF LIPASES IN SYNTHESIS OF STRUCTURED LIPIDS 349

reaction medium. Yoon and co-workers (Yoon et al., 1996) studied the effect of

pressure and temperature on the transesterification of triolein and ethylbehenate by

immobilized lipase Lipozyme IM. This enzyme is thermo stable in scCO

below



C (Marty et al., 1990; Chulalaksananukul et al., 1993) therefore the study

could be performed in the range of 40–70



C. The hydrolytic and transesterification

activity increased with increasing temperature. The increase in temperature enables

reactants to cross the activation energy barrier of the transition state (Whitaker,

1994). In scCO

the increase in temperature increases the solubility of triolein and

ethylbehenate and decreases the density. The lower density and viscosity of the

scCO

results in an increase in the mass transfer rate of the substrates and products

in the immobilized enzyme. The overall conversion reached its maximum between

50 and 60



The effect of pressure was also analyzed for the same system at 50



C. The

maximum transesterification activity was observed at 15 MPa while the hydrolytic

activity reached its maximum at 12 and 25 MPa. At lower pressures the rates of

both transesterification and hydrolysis decreased due to the low substrate solubility

at the temperature studied. The solubility of the substrates however increase with

increasing pressure at constant temperature (Chrastil, 1982) which may result in

the increase in the reaction rate. However, pressure increase can result in decreased

rates as reported for other systems (Erickson et al., 1990; Saito et al., 1994).

6.4. Chain Length

The chain-length substrate selectivity of various lipases has been studied in a

number of works (Janssen et al., 1996; Arsan et al., 2000). Among these, the

report by Vaysse and co-corkers (Vaysse et al., 2002) represents an exhaustive

description of the selectivity of various enzymes to different chain length substrates

for hydrolysis, esterification and alcoholysis in aqueous medium. scCo

was not

the reaction medium but it was shown that the transesterification of the triglyceride

with the ester of the fatty acid may be more effective than that with the free fatty

acid. This was attributed to the greatly decreased solubility of the free fatty acid in

with its increase in molecular weight (Yoon et al., 1996).

6.5. Continuous Synthesis

The continuous fixed bed reactor has been reported as being the most suitable

for development of new industrial applications e.g. ester synthesis by reversion of

hydrolysis (Balcão et al., 1996). The Lipozyme catalyzed esterification of oleic

acid with ethanol or the transesterification of triolein with ethanol in n-hexane in a

continuous packed-bed reactor showed a drastic loss of enzymatic activity with time

(Marty et al., 1997). This loss was attributed to the adsorption of polar substances,

water, or glycerol onto the enzyme support. Solutions advanced include the creation

of a hydrophilic layer that could prevent the diffusion of the hydrophobic substances

from the medium to the enzyme or the modification of the optimal thermodynamic