Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

Article abstract of DOI:10.1007/s11746-006-1245-4

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAC. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess > 99%) when an excess of ethanol was used. The result indicated that RML was highly stereosRMLelective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio = 31:1 and water content = 7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10:0), tridodecanoylglycerol (C12:0), tritetradecanoylglycerol (C14:0) and trioctadecenoylglycerol [triolein, (C18:1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used. Copyright

Full text of DOI:10.1007/s11746-006-1245-4

Enzymatic Preparation of Enantiomerically
Pure sn-2,3-Diacylglycerols: A Stereoselective
Ethanolysis Approach
Weera Piyatheerawonga, Tsuneo Yamaneb, Hideo Nakanoa, and Yugo Iwasakia,*
aLaboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan, and bDepartment of Environmental Biology,
School of Bioscience and Biotechnology, Chubu University, Aichi 487-8501, Japan
ABSTRACT: Stereoselective ethanolysis of monoacid TAG by DAG at high yield and purity was successfully achieved by di-
immobilized Rhizomucor miehei lipase (RML) was studied for
preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol
(TO) was used as a model substrate. The enantiomeric purity of
the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very
high (percent enantiomeric excess > 99%) when an excess of eth-
anol was used. The result indicated that RML was highly stereos-
elective toward the sn-1 position of TO under conditions of ex-
cess ethanol. The stereoselectivity of RML depended on the
amount of ethanol. The larger the amount of ethanol was, the
higher the stereoselectivity became. After optimizing the parame-
ters such as reactant molar ratio, water content, and temperature,
rect esterification of glycerol and FFA using 1,3-regiospecific
lipase (6–8) and by direct transesterification of glycerol and
vinyl esters of medium-chain FA using Candida antarctica li-
pase B (9).
In contrast to the preparation sn-1,3-DAG, chemoenzymatic
methods are used for the preparation of optically pure sn-1,2-
DAG or sn-2,3-DAG. Some recent publications describe syn-
theses of enantiomerically pure sn-1,2-DAG by lipase-medi-
ated sequential transesterification of the racemic O-alkyl glyc-
erols (10,11). In another synthetic route, the chiral sn-1,2-DAG
(ethanol/TO molar ratio = 31:1 and water content = 7.5 wt% of are chemically synthesized from (R)- and (S)-enantiomers of
the reactants at 25°C), optically pure sn-2,3-DO was obtained at
61.1 mol% in the glyceride fraction in 20 min. The above condi-
tions were further applied for ethanolysis of monoacid TAG with
different acyl groups such as tridecanoylglycerol (C10:0), trido-
decanoylglycerol (C12:0), tritetradecanoylglycerol (C14:0) and
trioctadecenoylglycerol [triolein, (C18:1)]. The yields and enan-
tiomeric purities of 1,2(2,3)-DAG were dramatically reduced
when TAG with FA longer than decanoic acid were used.
Paper no. J11267 in JAOCS 83, 603–607 (July 2006).
O-(4-methoxyphenyl)-glycidol, which are obtained from a one-
pot reduction of ketone and in situ lipase-mediated resolution
(12). Although the foregoing synthetic methods are accom-
plished by using an enzyme-catalyzed reaction as a part of the
process, the other parts mainly require chemical reactions,
which involve laborious steps and use of toxic reagents. A sim-
ple enzymatic process for the preparation of chiral sn-1,2- or
2,3-DAG has not been reported. One possible approach for this
purpose is lipase-catalyzed asymmetrical deacylation of
prochiral monoacid TAG at either the sn-1 or sn-3 position by
alcoholysis, which generates sn-2,3- or sn-1,2-DAG, respec-
tively. To establish an effective production method for chiral
sn-1,2- or sn-2,3-DAG, lipases with the desired stereoselectiv-
KEY WORDS: sn-2,3-Diacylglycerols, ethanolysis, monoacid
triacylglycerols, Rhizomucor miehei lipase.
DAG are widely used as emulsifiers in the food, cosmetics, and ity are required. Indeed, it is reported that some lipases discrim-
pharmaceutical industries. Frequently, mixtures of MAG and inate the sn-1 position from the sn-3 position of TAG in hydrol-
DAG are exploited because they are cheap and give appropri- ysis, interesterification, and ethanolysis (13–15).
ate performance (1). At present, DAG are regarded as healthful
In the present study, ethanolysis of trioctanoylglycerol (TO)
oils owing to their activity in preventing obesity (2). In addi- with immobilized Rhizomucor miehei lipase (RML) was ex-
tion, pure isomers of DAG have great potential as intermedi- amined as a reaction model. Several reaction parameters such
ates for the organic synthesis of phospholipids, glycolipids, as reactant molar ratio, water content, and temperature were in-
prodrugs, and structured lipids (3–6).
vestigated. Under the optimized reaction conditions, ethanoly-
DAG exist in three isomers: sn-1,3-, sn-1,2-, and sn-2,3- sis of monoacid TAG to prepare sn-2,3-DAG was demon-
DAG. For monoacid DAG, sn-1,2- and sn-2,3-DAG are enan- strated.
tiomers of each other. Although pure isomers of DAG are diffi-
cult to synthesize by traditional chemical synthetic methods, li-
pase-catalyzed regio- and stereoselective reactions would
EXPERIMENTAL PROCEDURES
provide alternative routes. For example, preparation of sn-1,3- Materials. TO, tridecanoylglycerol, tridodecanoylglycerol,
tritetradecanoylglycerol, and trioctadecenoylglycerol (triolein)
were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo,
Japan). Ethanol (99.5%), HPLC-grade n-hexane, and HPLC-
grade 2-propanol were purchased from Wako Pure Chemicals
*To whom correspondence should be addressed at Nagoya University, Furo-
cho, Chikusa-ku, Nagoya 464-8601, Japan.
E-mail: iwasaki@agr.nagoya-u.ac.jp
Copyright © 2006 by AOCS Press
603
JAOCS, Vol. 83, no. 7 (2006)

604
W. PIYATHEERAWONG ET AL.
(Osaka, Japan). Lipozyme RM IM (immobilized RML) was a The mobile phase was n-hexane/2-propanol (300:7) at flow rate
gift from Novozymes Japan Ltd. (Chiba, Japan). All other of 1.0 mL/min using an HPLC pump at 25°C. The peaks were
chemicals were of analytical quality or better.
detected using an ELSD. The drift tube temperature was 50°C.
Lipase-catalyzed ethanolysis. In a typical experiment, a re- Nitrogen was used as evaporation gas at 350 kPa. The weight-
action mixture consisting of 1.0 g TO, 3.0 g of ethanol, and based amount (in µg) of each enantiomer was calculated from
varying amounts of water (which was necessary to promote the the corresponding peak area using standard curves drawn with
reaction) was incubated at 35°C with stirring (500 rpm) for 15 known amounts of authentic 1,2-DAG (15). The enantiomeric
min to emulsify the mixture. The reaction was started by purity of 1,2(2,3)-DAG was evaluated from the enantiomeric
adding 0.4 g (10% of the reaction mixture) of immobilized excess (%ee) value, which was calculated using the weight-
RML. Portions (0.1 mL) of the reaction mixture were with- based amount of each isomer as follows;
drawn at intervals, mixed with 0.4 mL diethyl ether, and fil-
tered to remove the catalyst.
Analysis of the reaction mixture. The glyceride composi-
tions in the resultant lipid solution were analyzed with a
[sn - 2,3 - DAG]−[sn - 1,2 - DAG]
[sn - 2,3 - DAG]+[sn - 1,2 - DAG]
%ee =
×100
[1]
Analysis of the water content. The water contents in the re-
TLC/FID analyzer using Chromarod S III quartz rods (Iatron action mixtures were measured by a Karl-Fischer moisture
Laboratories, Tokyo, Japan). The rods loaded with the samples meter and expressed as the weight percentage of the reactants
were eluted for 10 cm with n-hexane/diethyl ether (9:1), and (lipids plus ethanol).
then the upper 8 cm was scanned for the quantification of FA
ethyl ester, TAG, and FFA. 1,3-DAG, 1,2(2,3)-DAG, 1(3)-
MAG, and 2-MAG, which remained close to the rod origin
RESULTS AND DISCUSSION
after the first development, were separated by a second devel- In the presence of an excess amount of ethanol, the lipase-cat-
opment with benzene/chloroform/acetic acid (100:5:1) and alyzed deacylation of prochiral monoacid TAG by ethanolysis
scanned for quantification.
initially yields chiral sn-1,2- and/or sn-2,3-DAG as intermedi-
Estimation of acyl migration. Nonenzymatic acyl migration ates, which are further converted to 2-MAG as the final prod-
from the sn-2 position to sn-1(3) of 1,2(2,3)-DAG or 2-MAG uct (16–18). Some lipases are known to have stereoselectivity
may form 1,3-DAG or 1(3)-MAG, respectively. The formed toward the sn-1 or sn-3 position in hydrolysis, interesterifica-
1,3-DAG or 1(3)-MAG are expected to be further deacylated tion, and ethanolysis (13–15).
by the 1,3-specific lipase, and eventually be converted to glyc-
Figure 1A demonstrates the typical time course for the
erol, decreasing the product yield. Since the concentration of change in glyceride composition during the ethanolysis of TO
1,3-DAG and 1(3)-MAG were very low throughout the reac- with immobilized RML at a 3:1 ethanol/TO weight ratio and at
tion, they would have been rapidly deacylated even if they had 0.8 wt% water content (no exogeneous water added). As the
been generated. Therefore, the amount of glycerol in the reac- reaction proceeded, TO was consumed, generating first the
tion mixture corresponds to the amount of glycerides lost 1,2(2,3)-dioctanoylglycerol [1,2(2,3)-DO] as the intermediate
through acyl migration followed by the complete deacylation. and then 2-monooctanoylglycerol (2-MO). The content of
Thus, the degree of the acyl migration can be evaluated from 1,2(2,3)-DO in the glyceride fraction reached approximately
the recovery of the glycerides, which can be calculated from 50% in 6 h. HPLC analysis of the 1,2(2,3)-DO revealed that
the amount of glycerol in the reaction mixture. The amount of the enantiomeric purity was absolutely to sn-2,3-DO (%ee >
glycerol was measured as follows. Sample solution (100 µL) 99%). The result was analogous to our previous report that
was transferred into a pre-weighed microcentrifuge tube, and RML greatly prefers the sn-1 position over the sn-3 position of
the solvent was evaporated completely under a nitrogen stream. TO in the presence of an excess amount of ethanol (15).
After weighing the residual materials, 100 µL of water was
Since the target product is an intermediate of the reaction, it
added to the tube. The mixture was briefly centrifuged, and the would be deacylated further to 2-MO, which is a by-product.
aqueous layer containing glycerol was recovered. The amount Thus, we focused on achieving both high yield and high enan-
of glycerol in the resulting aqueous solution was quantified by tiomeric purity of sn-2,3-DO. Figure 1B shows the influence of
an enzymatic assay using Glycerol Assay Kit (R-Biopharm ethanol/TO ratio on the yield and the enantiomeric purity of
AG, Darmstadt, Germany).
1,2(2,3)-DO. The stereoselectivity of RML was found to de-
Evaluation of enantiomeric purity. For investigating enan- pend on the amount of ethanol. The larger the amount of etha-
tiomeric purity of 1,2(2,3)-DAG, 40 µL of the sample solution nol was, the higher the stereoselectivity became. At an etha-
was evaporated at low temperature under vacuum and redis- nol/TO molar ratio of 31:1 (weight ratio = 3:1) or higher, the
solved in 40 µL of n-hexane. From the resulting lipid solution, %ee value was more than 99%. The yield of 1,2(2,3)-DO was
5 µL was injected onto the tandem column HPLC system (15), not affected very much by the reactant ratio, although the com-
in which a silica gel column (Ultrasphere 5 µm column, 4.6 × position of the other glycerides (i.e., the unreacted TO and the
250 mm; Beckman Coulter Inc., Fullerton, CA) and a chiral “over-deacylated” 2-MO) was different.
stationary phase column [CHIRALCEL ODTM cellulose-tris-
A possible explanation for the significant influence of etha-
3,5-dimethylphenyl carbamate-impregnated silica, 4.6 × 250 nol on the stereoselectivity of lipase could be related to the ef-
mm; Daicel Chemical, Tokyo, Japan) were connected in series. fect of solvent. In organic solvent, particularly water-removing
JAOCS, Vol. 83, no. 7 (2006)

SN-2,3-DIACYLGLYCEROLS AND STEREOSELECTIVE ETHANOLYSIS
605
FIG. 1. (A) Time course change of glyceride composition and enantiomeric purity of 1,2(2,3)-DAG during ethanoly-
sis of trioctanoylglycerol (TO). The reaction was performed at an ethanol/TO molar ratio of 31:1 (weight ratio of
3:1) and 35°C with no addition of exogenous water (water content was 0.8 wt% due to the endogeneous water).
(I) TAG (mol%), (L) 1,2(2,3)-DAG (mol%), (G) 2-MAG (mol%), (L) enantiomeric purity (%ee). (B) Influence of re-
actant ratio on glyceride composition and enantiomeric purity. The values shown are those determined when the
contents of the 1,2(2,3)-DAG were maximum.
solvent such as ethanol, the enzymes generally become more high temperature, suggesting that the active conformation of
rigid (19). In such an environment, the binding pocket of the the enzyme was not maintained even though it has water mole-
enzyme is relatively fixed to one geometry, resulting in highly cules in the system to stabilize its structure.
selective catalysis to a specific position of TAG.
Table 1 summarizes the ethanolysis of some other monoacid
Since the reactant molar ratio of 31:1 (weight ratio of 3:1) TAG of different chain lengths [tridecanoylglycerol (C10:0),
was somewhat better than the others with respect to the yield, tridodecanoylglycerol (C12:0), tritetradecanoylglycerol
further investigation was made at this reactant ratio. Water con- (C14:0), and trioctadecenoyl glycerol (C18:1)] as well as TO.
tent in the reaction is also an important parameter for this reac- The experiments were performed under the optimized condi-
tion system. As shown in Figure 2A, the reaction proceeded tions, i.e.; ethanol/TAG weight ratio = 3:1, water content = 7.5
slowly in a lower water-content environment, perhaps because wt% at 25°C. In the case of C10:0, the content of sn-2,3-DAG
ethanol strips off water molecules from the catalyst. Since most reached 58.7% in 50 min with high enantiomeric purity.
of enzymes require some amount of water molecules to main-
In contrast, in the cases of the other TAG having acyl groups
tain their active conformation, addition of exogenous water of C12 or longer, the accumulation level of 1,2(2,3)-DAG was
molecules could improve the reaction rate (20,21). As ex- very low. The reason for this very low accumulation of
pected, the reaction proceeded rapidly and selectively to give 1,2(2,3)-DAG is (i) deacylation of TAG was very slow and/or
sn-2,3-DO to approximately 60% among the glycerides in 30 (ii) deacylation of the generated 1,2(2,3)-DAG was too fast. In
min at a water content of 7.5 wt% or above (Fig. 2B). The re- the cases of TAG with C12 or C18:1, RML deacylated 1,2(2,3)-
sult was in accordance with our previous report indicating that DAG rapidly to 2-MAG, accumulating smaller amounts of
RML needs a certain amount of water molecules to perform the 1,2(2,3)-DAG and larger amounts of 2-MAG. TAG with C14
catalytic reaction (18). When the water content was 4.8 wt% or was not ethanolized very much, and the 1,2(2,3)-DAG gener-
above, small amounts of FFA were detected because of hydrol- ated was quickly deacylated, resulting in little accumulation of
ysis. The amount of the FFA was less than one-tenth that of the 1,2(2,3)-DAG.
FA ethyl esters throughout the reaction, indicating the domi-
nant reaction that took place was not the hydrolysis but the glycerol formed was more than 80 mol% for all the different
ethanolysis. TAG tested. Considering the recovery of the glyceride, the
The recovery of the glyceride estimated from the amount of
Reaction temperature is another key parameter for the reac- yield of 1,2(2,3)-DAG is 54.0 and 50.5 mol% for the cases of
tion. As illustrated in Figure 3, the reaction progressed rapidly C8:0 and C10:0, respectively, which is satisfactory for practi-
at lower temperatures, and the highest content of sn-2,3-DO cal purposes.
(61.1%) was achieved in only 20 min at 25°C with high enan-
Glyceride recovery of more than 80 mol% also indicates
tiomeric purity. By contrast, the reaction proceeded slowly at that certain portions (less than 20 mol%) of the glyceride were
JAOCS, Vol. 83, no. 7 (2006)

606
W. PIYATHEERAWONG ET AL.
FIG. 2. (A) Comparison of 1,2(2,3)-dioctanoylglycerol [1,2(2,3)-DO] formation during ethanolysis reaction with dif-
ferent water contents. The reaction was performed at an ethanol/TO molar ratio of 31:1 (weight ratio of 3:1) and
35°C. (I) 0.8 wt%, (L) 2.9 wt%, (G) 4.8 wt%, (I) 6.7 wt%, (L) 7.5 wt%. (B) Influence of water content on glyc-
eride composition and enantiomeric purity. The values shown are those determined when the contents of the
1,2(2,3)-DAG were maximum. For other abbreviation see Figure 1.
lost as glycerol by complete deacylation. Since RML is known were detected throughout the reaction, the deacylation of 1,3-
to be very strictly 1,3-position specific, it is unlikely that the li- DAG and 1(3)-MAG may be much faster than acyl migration
pase was able to attack the glycerides randomly at the sn-2 po- itself. From the above consideration, the amount of glycerol (=
sition as well as the sn-1(3) positions. Therefore, the complete loss of the glyceride) can be used as a measure to estimate the
deacylation should involve nonenzymatic acyl migration from degree of acyl migration. Judging from the amounts of glyc-
the sn-2 position to sn-1(3) position in 1,2(2,3)-DAG or in 2- erol (less than 20 mol%), the degree of acyl migration under
MAG to generate 1,3-DAG or 1(3)-MAG, respectively. The the reaction conditions is not so high as to reduce the yield of
generated 1,3-DAG and 1(3)-MAG are the substrates of RML product seriously.
and are thereby further deacylated to glycerol. Since no 1,3-
The present study provides a simple enzymatic method for
DAG and only a very small amount of 1(3)-MAG (<1 mol%) preparation of sn-2,3-DAG with an environmentally friendly
FIG. 3. (A) Comparison of 1,2(2,3)-DO formation during ethanolysis reaction at various temperatures. The reaction
was performed at an ethanol/TO molar ratio of 31:1 (weight ratio 3:1) and a water content equal to 7.5 wt%. (I)
25°C, (G) 35°C, (L) 40°C, (I) 50°C. (B) Influence of temperature on glyceride composition and enantiomeric pu-
rity. The values shown are those determined when the contents of the 1,2(2,3)-DAG were maximum. For abbrevia-
tions see Figures 1 and 2.
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SN-2,3-DIACYLGLYCEROLS AND STEREOSELECTIVE ETHANOLYSIS
607
TABLE 1
Summary of Rhizomucor miehei Lipase-Catalyzed Ethanolysis of TAG with Various Acyl Groupsa
Selectivity
and %ee of
Glyceridec (mol%) 1,2(2,3)-DAG
Glyceride composition (mol%)b
Recovery of
TAG (chain length)
Time (min)
TAG
1,2(2,3)-DAG
2-MAG
Trioctanoyl glycerol (C8:0)
20
50
420
540
360
17.8
11.5
1.8
68.2
17.9
61.1
58.7
7.2
1.3
25.0
21.1
29.8
90.9
30.5
57.1
88.4
86.2
81.4
87.4
86.6
sn-1, >99%
sn-1, >99%
NDd
Tridecanoyl glycerol (C10:0)
Tridodecanoyl glycerol (C12:0)
Tritetradecanoyl glycerol (C14:0)
Trioctadecenoyl glycerol (C18:1)
NDd
sn-1, 67%
aThe reactions were performed at 25°C, ethanol/TAG weight ratio of 3:1, and water content of 7.5 wt%.
bComposition of the reaction mixture when the contents of 1,2(2,3)-DAG were maximum.
cRecovery of glycerides [= TAG, 1,2(2,3)-DAG and 2-MAG] was estimated from the amount of the glycerol.
dNot determined due to the very low amount of 1,2(2,3)-DAG.
10. Guanti, G., L. Banfi, A. Basso, E. Bevilacqua, L. Bondanza, and
R. Riva, Efficient Chemoenzymatic Enantioselective Synthesis
of Diacylglycerols (DAG), Tetrahedron Asymmetry
15:2889–2892 (2004).
approach. Since sn-2,3-DAG is the building block for various
organic compounds, it will be useful as a potential intermedi-
ate in organic synthesis.
11. Halldorsson, A., P. Thordarson, B. Kristinsson, C.D. Magnus-
son, and G.G. Haraldsson, Lipase-Catalyzed Kinetic Resolution
of 1-O-Alkylglycerols by Sequential Transesterification, Ibid.
15:2893–2899 (2004).
12. Kamal, A., M. Sandbhor, A.A. Shaik and M.S. Malik, A Facile
and Convenient Chemoenzymatic Synthesis of Optically Active
O-(4-Methoxyphenyl)-glycidol and 1,2-Diacyl-sn-glycerol,
Ibid. 16:1855–1859 (2005).
ACKNOWLEDGMENTS
The authors are grateful to Novozymes Japan Ltd. (Chiba, Japan)
for the generous supply of the immobilized lipases. The Ministry of
Science and Technology (MOST, Thailand) is acknowledged for fi-
nancial support to W. Piyatheerawong.
13. Rogalska, E., C. Cudrey, F. Ferrato, and R. Verger, Stereoselec-
tive Hydrolysis of Triglycerides by Animal and Microbial Li-
pases, Chirality 5:24–30 (1993).
14. Iwasaki, Y., M. Yasui, T. Ishikawa, R. Irimescu, K. Hata, and
T. Yamane, Optical Resolution of Asymmetric Triacylglycerols
by Chiral-Phase High-Performance Liquid Chromatography, J.
Chromatogr. A 905:111–118 (2001).
15. Piyatheerawong, W., Y. Iwasaki, and T. Yamane, Direct Sepa-
ration of Regio- and Enantiomeric Isomers of Diacylglycerols
by a Tandem Column High-Performance Liquid Chromatogra-
phy, J. Chromatogr. A 1068:243–248 (2005).
16. Irimescu, R., K. Furihata, K. Hata, Y. Iwasaki, and T. Yamane,
Utilization of Reaction Medium-Dependent Regiospecificity of
Candida antarctica lipase (Novozym 435) for Synthesis of 1,3-
Dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) Glycerol,
J. Am. Oil Chem. Soc. 78:285–289 (2001).
17. Irimescu, R., Y. Iwasaki, and C.T. Hou, Study of TAG Ethanol-
ysis to 2-MAG by Immobilized Candida antarctica Lipase and
Synthesis of Symmetrically Structured TAG, Ibid. 79:879–883
(2002).
18. Piyatheerawong, W., Y. Iwasaki, X. Xu, and T. Yamane, De-
pendency of Water Concentration on Ethanolysis of Trioleoyl-
glycerol by Lipases, J. Mol. Catal. B–Enzym. 28:19–24 (2004).
19. Schmitke, J.L., C.R. Wescott, and A.M. Klibanov, The Mecha-
nistic Dissection of the Plunge in Enzymatic Activity upon
Transition from Water to Anhydrous Solvents, J. Am. Chem.
Soc. 118:3360–3365 (1996).
20. Yamane, T., Importance of Moisture Content Control for Enzy-
matic Reactions in Organic Solvents: A Novel Concept of “Mi-
croaqueous,” Biocatalysis 2:1–9 (1988).
21. Klibanov, A.M., Why Are Enzymes Less Active in Organic Sol-
vents Than in Water? Trends Biotechnol. 15:97–101 (1997).
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[Received November 2, 2005; accepted May 12, 2006]
JAOCS, Vol. 83, no. 7 (2006)