Biotechnology Advances. Vol. 15, No. 1. pp. l-15.1997 Cowrieht 0 1997 Elsevier Science Inc. Rinteiin the USA. All rights reserved

0734-9750/97 $32.00 + 00

ELSEVIER PII SO734-9750(96)00046-S

ENZYME CATALYZED ESTERIFICATION

ROBERT LORTIE

Bioprocess Research and Development Biotechnology Research Institute, National Research Council of Canada

6100 Royalnwunt Avenue, Montrt_!al, Q&bee. H4P 2R2, Canada

ABSTRACT

Enzyme catalyzed esteritication reactions have found many applications, ranging from the modification of vegetable oils for human consumption to the production of optically pure chemicals, To displace the equilibrium in favor of synthesis, rather than hydrolysis, these reactions are performed in non-aqueous or microaqueous media. The influence of the amount of water, and of the nature of organic solvent, are new parameters to consider in the optimization of industrial processes. They also add a new perspective to our knowledge of the functioning of enzymes. e 1997 Ekcvicr S&ace Inc.

KEY WORDS

Enzyme catalysis, esteritication, lipase, esterase, chiral production.

INTRODUCTION

Like other catalysts, enzymes affect rates of reversible reactions in both directions [39]; the direction

taken is determined by thermodynamics. In nature, oxydoreductases perform both oxidation and

reduction reactions, depending on the relative concentrations of electron accepting and donating

species, and it is possible to displace the equilibrium by electrochemically regenerating nicotinamide

cofactors [45]. The displacement of the equilibrium in reactions catalyzed by simple enzymes such as

hydrolases is achieved by changing the concentration of the reactants, the main one being water. By

diminishing the concentration or activity of water, it is theoretically possible to use any hydrolase to

catalyze the corresponding synthetic reaction.

This article is dedicated to the memory of my father, Gilles Lortie (1930-1996)

2 R. LORTIE

Many strategies have been utilized to displace the equilibrium. Water miscible solvents or

water activity repressing agents such as salts and polyols have been used, and enzymes have been

deployed in water-free or almost water-free media-solvents, pure liquid substrates, and the gas

phase. Various enzymes have been studied in these systems, and the role of factors such as the nature

of the solvent and the concentration or thermodynamic activity of water have been examined The

major advances are reviewed here with a focus on enzymatic production of esters. This class of

molecules is present in fats and oils, and natural and synthetic polymers. Esters are also useful

intermediates or end products in the chemical industry. Various hydrolases-for example, esterases,

lipases and proteases-may act on ester bonds, depending on the conditions and the nature of the

substrate. This subject continues to be a focus of considerable attention as reflected by the

presentations at a recent symposium on biotransformations in non-conventional media: more than

half of the papers dealing with enzymes discussed esterolytic and lipolytic enzymes [82].

BEHAVIOR OF ENZYMES IN SYNTHETIC CONDITIONS

Enzymes may be used under two different states in non-conventional media: in solution or as solids.

In the first case, they can be in the native form, or modified with amphiphilic molecules to improve

their solubility in organic or water/organic media. Solid enzymes may be used as precipitates,

liophylizates, cross-linked crystals, or immobilized. These enzymes have been reported to follow the

same bi-bi ping-pong mechanism-acyl-enzyme intermediate and competitive inhibition by the

nucleophile-in the synthetic reaction as in the hydrolytic one [I 5,18,20,54]. In all these cases,

parameters not relevant in aqueous solutions-concentration of water and the nature of any

solvent-may be of great importance [5].

Role of water

Water has many different and possibly contradictory roles in these systems. To favor synthesis, the

thermodynamic activity of water should be kept as low as possible. However, water has an important

role in the three dimensional structure of proteins, in solution as well as in the solid state, and hence

on catalytic tmtction [81]. It was recognized very early that the amount of water present was

important for both the thermal stability [88] and the activity [95] of enzymes in non-aqueous media.

Being the nucleophile in the hydrolysis reaction, water can also be a competitive inhibitor, as

reported for lipase-catalyzed synthesis of dodecyl decanoate in hexane [85]. Although the activity of

ENZYME CATALYZED ESTERIFICATION 3

Rhrzomwor miehei lipase used in the synthesis of dodecyl decanoate varies from one solvent to

another, the maximum activity in different solvents remains the same for a given value of water

activity, CIW. When the catalytic activity is reported in terms of molar concentration of water, the

optimal concentration depends on the solvent [84]. It has also been shown that R. miehei and

Candida rqpsa lipases immobilized on various supports show similar dependence of their activity

on nw [86], whereas the activity of subtilisin depends strongly on the nature of the support for

constant total water content [63]. If one considers that the amount of water adsorbed onto solid

proteins as a fimction of water activity is the same in air and in various organic solvents at low water

activities values [35,56], and the fact that it is the amount of enzyme-bound water that determines

the activity of the enzyme [95], it is then obvious that the best way to take the water content into

account is to use the thermodynamic water activity. This has also been observed in solid/gas catalysis

involving cutinase and lipase [44] as well as other enzymes [92,93].

Role of the solvent

The influence of the nature of the solvent has been studied extensively and interpreted in terms of

various phenomena. Changes in the rigidity of the enzyme caused by the solvent dielectric screening

of the polar and ionic interactions in the protein have been pointed out [2,3]. The stabilization of

charged transition states through modification of the active site polarity [90], as well as variations in

to the total free energy change associated with the different solvation energies of solvents are other

possible causes of the solvent effect [71]. No clear consensus has yet emerged on the choice of

parameter for quantitatively describing the effect of solvents on enzyme reactions, The most

frequently used is parameter is log P, where P is the partition coefficient of the solvent between

I-octanol and water. However, log P has been reported to not correlate well with enzyme efficiency

[61], and, therefore, other parameters such as dielectric constant [2,3], polarizability and electron

acceptance index [87] as well the Hildebrand solubility parameter [14] and a three dimensional

solubility parameter derived from it [75] have been proposed. In a recent article, Schmitke et al. [74]

were able to breakdown the dramatic loss of activity of cross-linked crystals of subtilisin Carlsberg,

for the transesterification of N-Ac-L-Phe-OEt with propanol in acetonitrile, into three different

contributions: the change in pH profile caused by the reticulation, the increased stability of the

solvated state of the substrates in acetonitrile, and the increased rigidity of the enzyme molecule at

low water activity.

4

Alcoholysls

P R-C-O-R,’ 3

Acidolysis

ti P t P R,-C -0 -R’ .I R,-C-O-H - R,-C-O-R’ + R,-C-O-H

t fl ii P R,-C -0 -R,‘+ R,-C-O -R,’ - R,-C -0 -R,’ + R,--C-O-R,’

R. LORTIE

:: OH-R,’ p R-C-O-R,’ + OH-R,’

Figure 1. Transesterification reactions,

APPLICATIONS

Lipases as a class of enzymes have experienced the greatest increase in their market share during the

past few years [89]. One major reason for this is the development of heat stable lipases such as

Novo’s Lipolase, to enhance the fat and oil cleaning capacity of laundry detergents. Non-hydrolytic

applications have also been developed. They cover a wide spectrum of molecules and applications.

The production of esters can be achieved either by synthesis from free acid and hydroxyl groups or

by ester exchange or transesterification. Transesteritication includes alcoholysis, acidolysis, and

interesterification [91]. These reactions are summarized in Figure 1. Aminolysis is also a

transesteritication reaction, but the production of amide bonds is beyond the scope of this article. All

these reactions proceed through the formation of the acyl-enzyme intermediate followed by

nucleophilic attack. The biggest difference lies in the fact that in acidolysis and interesterification, a

hydrolytic reaction must take place first to free a nucleophile which will then attack the acyl-enzyme

formed with the acyl moiety of the new ester to be produced [48,67], adding a new dimension to the

importance of water.

ENZYME CATALYZED ESTERIFWATION

Fats and oils modification

Some of the most important esters are the glycerides present in the fats and oils used in food and

other industries, Enzymes, especially lipases, have many possible uses in treatment and modification

of fats and oils [59]. The recent advances in food sciences, showing the importance of unsaturated

fatty acids and the danger of cis to /KW isomerization during chemical hydrogenation of vegetable

oils [S7] have fostered the development of bioprocesses employing milder conditions for the

preparation of vegetable shortenings and margarines [50]. By exchanging some fatty acids, totally or

in part, of the triglycerides of a given vegetable oil with those from a different oil, it is possible to

modify the physico-chemical and nutraceutical properties, and palatability of the starting material

[32,40,96].

Processes employing solvent free media [27,28,33] as well as organic solvents [12,50] have

been studied. Major effort has also been aimed at production of a substitute for cocoa butter using

cheaper vegetable oils [ 10,47,55,58]. In this instance, the selectivity of most lipases for the 1 and 3

positions on the glycerol backbone has been found to be advantageous, because most important

triglycerides present in cocoa butter have palmitic or stearic acid at positions 1 and 3, and oleic acid

strictly at position 2, and chemically catalyzed interesteritication is not selective [65]. It should be

noticed that even though it is possible to obtain mono- and diglycerides with high selectivity [6,7],

the enzyme’s specificity can be hampered by acyl migration, and that it is possible to produce

triglycerides from glycerol and free fatty acids using a 1,3-specific lipase [ 11,29,46]. It is also

possible to produce triglycerides enriched in long chain fatty acids of fish oils [36,79] which are

helptul in preventing coronary diseases [34]. In this instance, the specificity of some lipases against

given fatty acids, and especially long chain o-3 polyunsaturated ones, is helpful. However, it should

be stressed that the specificity determined in hydrolysis reactions is not always maintained in

synthetic conditions [78]. Other interesting products are the “structured lipids” [40] in which

medium chain fatty acids are present at positions 1 and 3, and long chain polyunsaturated ones at

position 2. These lipids are easier to digest and can still provide essential fatty acids They are

especially suited for infant food formulations and for patients with absorption problems.

Other possible applications of lipase interesteritication are in easing handling of palm oil by

lipase catalyzed interesteritication with canola or soybean oil in order to decrease the low

temperature viscosity of the oil [43]. Lipase catalyzed esteritication can be applied to the enzymatic

refining of hyperacid oils by esterification of their free fatty acids with [8,26] or without added

glycerol [8,42]. It is possible to prepare wax esters, that can be used in personal care products,

6 R. LORTIE

through direct synthesis from free fatty acids and long chain alcohols or by alcoholysis of

triglycerides [83]. Enzymatic modification of vegetable oils through transesterification has already

been implemented at the multi-ton scale: Loders Croklaan has built a plant for that purpose at

Wormeveer in the Netherlands [66]

Chirrl production

During the last decade, it has become increasingly important to consider enantiomers of a given

molecule as two different entities in terms of their biological action, especially pharmacological

properties [4]. The US Food and Drug Administration has issued guidelines concerning the

development of drugs having chiral centers [30]. Among the different methods available, kinetic

resolution of racemates or asymmetrical synthesis from prochiral precursors, both enzyme catalyzed,

are interesting [22,72,77]. The first generation of processes developed was based on the hydrolysis

of derivatives such as acylated amines or esterified alcohols and acids. In recent years,

enantioselective synthesis and transesterification reactions performed in organic media have been

extensively studied and described [41,5 I]. These reactions have many advantages over hydrolysis.

For instance, solubility problems can be overcome more easily than in aqueous solutions, and the

number of steps can be reduced, since prior derivatization may not be necessary. Increased stability

of enzymes in organic solvents has been reported [88,94], as well as increased stereoselectivity in the

synthetic mode [ 161. In any kinetic resolution process, the enantioselectivity comes from the ratio of

the rates of reactions of the two enantiomers present It is therefore certain that the nature of the

solvent and the water content may have an effect on the enantioselectivity, because the two reactions

may be affected differently. Indeed, changes in stereoselectivity, and even stereoselectivity reversal

[SO], caused by changes in solvent or water content have been reported [32,37]. The lack of

influence of water activity has also been reported [ 131

Polymer production

Because of the high selectivity of enzymes, it is possible to produce polymers with high degrees of

chemical, regio or optical selectivity [23]. For instance, lipases have been used to synthesize optically

active polymers from a racemic mixture of monomer [52]. Protease catalyzed esterification of

sucrose and bis(2,2,2-trifluoroethyl)adipate led to a water-soluble polymer with no detectable

crosslinking, despite the presence of eight hydroxyl groups on the disaccharide [64]. Similarly,

lipase-catalyzed esterification of various monosaccharides with vinyl acrylate produced the 6-acryloyl

ENZYME CATALYZED ESTERIFICATION 1

esters which gave, upon chemical polymerization, water soluble polyacrylates that could be

crosslinked to obtain insoluble material with the capacity to absorb up to SO-fold its weight of water

1531.

Esterification of sugars and sugar alcohols

Monoesters of fatty acids and sugars or sugar alcohols constitute a very interesting group of non-

ionic surfactants with potentially important applications in many industries. These types of

amphiphilic molecules have very good emulsifying, stabilizing or conditioning effects. These

products are difficult to obtain by standard high temperature chemical esterification which causes

coloration of the final product and dehydration and cyclization in the case of sugar alcohols. The use

of a biological catalyst under mild conditions for the synthesis of esters of sugars or sugar alcohols

can overcome these problems and enzymatic approaches have been developed in recent years [69].

The synthesis of fatty acid sugar esters in aqueous media has been reported [38,76], but this method

has a low yield. Klibanov and coworkers have synthesized acylated sugars in organic solvents in

which both sugars and fatty acids are soluble [17,68]. The lipase-catalyzed preparation of alkyl

glucoside fatty acid esters was made in a benzenelpyridine mixture [60] or in solvent-free media

[ 1,9]. Since solubility of many sugars in non-polar solvents is very low, it has been proposed to use

organoboronic acids which are known to form carbohydrate-boronate complexes by reversible

condensation with carbohydrates. In this case, yields obtained in closed vials are noticeably increased

[62,73] but there are at least two additional steps. The use of bulky tertiary alcohols as solvent

combined with an efficient removal of the water under reduced pressure has also been described.

Conversions and yields were very good [24] and the molecules had good tensioactive properties

P51.

Short chain esters

Short chain esters are important constituents of natural and artificial flavors and fragrances [70].

Because of the mild reaction conditions required. enzyme catalysis is well suited for the production

of these molecules. Lipase-catalyzed esterification and transesterification of terpene alcohols have

been performed in solvents [ 18,211 and in supercritical carbon dioxide [ 191. Esters of alkyl alcohols

and alkyl acids were also produced in solvents [49] and in gas/solid reactors [44]. This last case,

where the enzyme catalyst is present in the solid form and the substrates and products are in the

gaseous state is especially appropriate for these highly volatile compounds. This technology, as well

8 R. LORTIE

as the use of supercritical carbon dioxide, offer an interesting, environment friendly alternative to the

use of organic solvents for non-aqueous enzymology.

CONCLUSION

Enzyme catalyzed production of esters offers interesting avenues at the lab scale as well as at

industrial scale. The mild conditions allow transformation of labile substrates; regio and

stereoselectivity of enzymes permits production of optically pure derivatives in high yield and

without the need for expensive separation procedures.

Acknowledgment

The author wishes to thank Dr. Amelie Ducret for helpml discussions

1.

2.

3.

4

5.

6.

7.

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