Coprecipitation of Pseudomonas fluorescens lipase with hydrophobic compounds as an approach to its immobilization for catalysis in nonaqueous media

Article abstract of DOI:10.1023/A:1013902202789

The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone-water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of th

Full text of DOI:10.1023/A:1013902202789

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 1, 2002, pp. 38–43. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 1, 2002, pp. 44–49.
Original Russian Text Copyright © 2002 by Gorokhova, Ivanov, Zubov.
Coprecipitation of Pseudomonas fluorescens Lipase
with Hydrophobic Compounds As an Approach
to Its Immobilization for Catalysis in Nonaqueous Media
1
I. V. Gorokhova, A. E. Ivanov, and V. P. Zubov
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
Received November 27, 2000; in final form, March 11, 2001
Abstract—The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and
poly(butyl metacrylate) in acetone–water media in the presence of the lipase from Pseudomonas fluorescens
was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the
lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation
with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be
arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cety-
lacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. Immobilization of the
lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the
presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme.
Key words: cetyl alcohol, ester synthesis, hexadecane-1,2-diol, immobilization, N-cetylacetamide, poly(butyl
metacrylate)
1
INTRODUCTION
inclusion into hydrophobic gels [11] and hydrophobic
coprecipitates [12], and treatment of the enzymes with
synthetic lipid-like agents [13], are thus studied inten-
sively. Numerous immobilization methods ensure that
we obtain catalysts with wide-ranging characteristics.
For the preparations of lipases immobilized on a num-
ber of solid supports, the highest initial rate of alcohol
esterification in a model reaction (6- to 7-fold higher
than that for the nonimmobilized enzyme) was
observed in the case of the enzyme adsorption on Celite
The enzyme-catalyzed enantioselective synthesis of
organic compounds has recently become an intensively
developed field of bioorganic and organic chemistry.2
This is dictated by the necessity to have a wide assort-
ment of chiral organic compounds of high optical
purity, including some medicines and their components
and various biochemical agents. Lipases (EC 3.1.1.3)
occupy a special place in the series of hydrolytic
enzymes used as catalysts for many transformations in
organic chemistry, as they can be used both for the
hydrolysis of lipids and esters and for the enantioselec-
tive esterification and transesterification in organic sol-
vents or water–organic media [1–7]. Factors like strict
stereospecificity and the high rate of the enzymatic
5
45 particles, whereas the operational stability (the
enzyme activity after ten working cycles) was higher in
the case of the enzyme covalent binding to the Eupergit
C250L support [10].
We demonstrated previously that the incorporation
reactions and the possibility to perform these processes of lipases into hydrophobic coprecipitates with HDD
under mild conditions favor the industrial use of [12] increases significantly (more than tenfold) the cat-
lipases. However, in industry, only the use of immobi- alytic enzyme activity in the enantioselective esterifica-
lized enzymes is highly profitable, because this makes tion. A marked increase in the enzymatic activity is also
the separation from reaction systems and repeated use observed upon enzyme treatment with lipid-like
of the enzymes much easier.
Various approaches to lipase immobilization, such
as physical sorption on hydrophobic adsorbents [8–10],
reagents [13]. However, these immobilization methods
are still poorly investigated. Hence, the study of the
compounds structurally similar to HDD and also some
polymers with similar solubilities and the balance of
hydrophilic and hydrophobic properties is of interest.
1
Please address correspondence to: phone. +7 (095) 336-0600,
e-mail: irinagorokhova@ibch.ru.
In this study, we focused on lipase inclusion into
coprecipitates with a number of hydrophobic com-
pounds, developed approaches to the preparation of
recoverable catalysts on the basis of the Celite 545 and
2
Abbreviations: AIBN, azoisobutyronitrile; CeAA, N-cetylacet-
amide; CeNH , N-cetylamine; CeOH, cetyl alcohol; HDD, hexa-
2
decane-1,2-diol; PBMA, poly(butyl metacrylate); and TBME,
t-butyl methyl ether.
1
068-1620/02/2801-0038$27.00 © 2002 MAIK “Nauka /Interperiodica”

COPRECIPITATION OF PSEUDOMONAS FLUORESCENS
39
O
O
CH COCH CH
OH
OH
O
CH3
Lipase
CH CHO
+
+
3
2
–
3
Ph
Ph
CH3
CH3
Ph
R-(II)
CH3
RS-(I)
S-(I)
The scheme of the enzymic esterification of (1RS)-phenylethanol with vinyl acetate.
Eupergit C250L solid particles, and suggested a modi- between the positively charged ëÂNH particles and
2
fied procedure for measuring the rate of the model reac-
tion: enantioselective acetylation of (1RS)-phenyletha-
nol (RS-(I)) with vinyl acetate in the TBME medium
the negatively charged lipase molecules. The efficiency
of the lipase incorporation into the precipitates
decreases in the series of coprecipitants: ëÂNH –
2
(see scheme).
D
.7
RESULTS AND DISCUSSION
0
The Composition of the Reaction Mixture
in the Model Reaction
(a)
0.6
0.5
The pure product of enzymatic acetylation, (1R)-
phenylethyl acetate (R-(II)) (scheme) was isolated by
HPLC instead of gas chromatography, which was used
previously [10]. Under standard conditions of rpHPLC
0
.4
0.3
(
elution with 1 : 1 water–acetonitrile isocratic system),
0
0
.2
.1
more hydrophobic compounds are retained stronger by
the sorbent with octadecyl groups. As hydrophobicity
increases in the order (I), vinyl acetate, and (II), the
retention times for these compounds increase in a sim-
ilar order and are 5.9, 6.2, and 13.5 min, respectively
0
.4
(Fig. 1). The content of the product (II) in the reaction
(b)
mixture was determined from the square of its chro-
matographic peak in the optical density–time coordi-
nates taking into account its known molar extinction
coefficient ε260 (80 å cm ). The correlation between
the content of (II) measured by this method and deter-
mined by GC showed the difference to be less than 3%.
0.3
0.2
–1
–1
0
.1
Preparation and Properties of the Lipase
Coprecipitates with Hydrophobic Compounds
Coprecipitates of the lipase with hydrophobic com-
pounds were obtained as described for the lipase copre-
cipitate with HDD [12]. The lipase coprecipitants with
the same length of hydrophobic moiety (C ), but vary-
ing in the terminal functional group, as well as PBMA,
were used. The enzyme content in the coprecipitates
was calculated from its residual activity in the superna-
tant:
(c)
0
0
.3
.2
1
6
0.1
lipase incorporation into precipitate (%)
lipase activity in supernatant
2
4
6
8
min
10 12 14


=
1 – ------------------------------------------------------------------ -- × 100.


starting lipase activity
Fig. 1. The rpHPLC analysis of the reaction mixture upon the
enantioselective acetylation of (1RS)-phenylethanol with
vinyl acetate catalyzed by the native lipase. The reaction time:
(a) 0, (b) 24, and (c) 97 h; conversion of (1RS)-phenylethanol:
(b) 25 and (c) 50%.
The maximal enzyme inclusion (more than 99%)
into the coprecipitates was observed upon coprecipita-
tion with ëÂNH (Table 1). This effect can be explained
2
by the contribution of the electrostatic interaction
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

4
0
GOROKHOVA et al.
Table 1. Enzymic activity of the native lipase and its copre- of the phenylethanol (RS-(I)) acetylation with vinyl
cipitates
acetate catalyzed by various lipase coprecipitates
Table 1). The esterifying activity of the enzyme copre-
(
Activity*
cipitates decreases in the series of the coprecipitants
Enzyme
inclusion in co-
precipitate, %
Lipase
coprecipitant
in (1RS)-phe-
nylethanol
acetylation
CeAA–ëÂNH –HDD–PBMA–CeOH. Note that the
2
in triacetin
hydrolysis
catalysts active in organic solvent are less active in
aqueous medium (Table 1). This may be due to the dif-
ferent wettability of the coprecipitates and also to the
differences in their structural organization that affects
the transport of substrates to the lipase active sites. At
the same time, the marked difference in the activity of
the coprecipitates in esterification suggests that more or
less specific binding of the lipase to coprecipitants
exists. Taking into consideration the equal or very close
lengths of the hydrophobic substituents in the coprecip-
itants (except for PBMA), one can assume that the
binding is dictated by polar interactions (electrostatic
interactions and hydrogen bonding) with the coprecipi-
–
–
435
200
250
490
150
46
1530
19000
13600
9300
CeAA
CeNH2
HDD
65
99
85
80
90
CeOH
2500
PBMA
2900
*
In (µmol of product) h–1 (mg of enzyme)–1.
PBMA–HDD–CeOH–CeAA. However, in the case of tants, which result in the most active coprecipitates
CeOH, the supernatant was found to contain very small (CeAA and ëÂNH ). It should be noted that all the
2
particles of the coprecipitate that do not sediment under obtained coprecipitates could be isolated from reaction
the conditions used for its isolation. Therefore, the mixtures and, hence, used repeatedly. This fact is
enzyme content in this coprecipitate (80%) is only ten- important from practical point of view, and, in this
tative. Another regularity is observed upon the determi- regard, PBMA offers the greatest promise as a copre-
nation of the catalytic activity of coprecipitates for tri- cipitant, since its coprecipitates with the lipase can
acetin hydrolysis (Table 1). The hydrolytic activity of most easily be isolated from both the aqueous medium
the lipase does not remain intact: the activity falls in the and TBME. Therefore, the use of polymeric coprecipi-
series of coprecipitants HDD–ëÂNH –CeAA–CeOH– tants could create new methods for obtaining recover-
2
PBMA. Some activation of the lipase (1.13-fold) is able catalysts. In addition to high catalytic activity, the
observed only in the case of its coprecipitate with cheapness of the starting materials is one of the advan-
HDD. The stabilization of lipases by cis-diols (e.g., sor- tages of the resulting coprecipitates and profitably dif-
bitol) was observed [14], and this may be the reason for ferentiates them from the previously suggested lipid-
the high activity of the enzyme in its coprecipitates with like agents, which are difficult to obtain [13].
HDD.
The most interesting results were obtained upon the
The Enantioselectivity of Esterification
determination of the activity of the coprecipitates in a
model esterification reaction in an organic medium
The study of the optical activity of the samples of
(
Fig. 2). The growth of enzymatic activity as compared
1
-phenylethyl acetate obtained by esterification cata-
to that of the native enzyme was observed in all cases
lyzed by the lipase and its coprecipitates testified to the
retaining of the enantioselectivity of esterification
Table 2). For all of the coprecipitates, the values of
(
Yield of (II),%
specific optical rotation are of the same sign and fall
within the same range (70°–80°). The differences in the
optical rotation values are due to the product isolation
at various degrees of the RS-(I) to R-(II) conversion.
Since the acetylation of the R-form of the alcohol is
accompanied by the slow acetylation of its S-form, the
optical rotation of the product decreases with an
increase in the RS-(I) conversion. Comparative experi-
ments with the native lipase and the coprecipitate of the
lipase with CeOH (Table 2) clearly demonstrated this
statement. More than 99% enantiomeric purity of
R-(II) was observed in the case of the catalysis by the
native lipase at 48% conversion of RS-(I) [7]. Based on
the data represented, one can conclude that the enanti-
omeric purity of the product in the model reaction
remains high upon the use of the lipase coprecipitates
as catalysts.
5
4
3
2
1
0
0
0
0
0
2
4
6
5
1
3
0
5
10
15
20
25
30
h
Fig. 2. The kinetic curves of 1-phenylethyl acetate accumu-
lation in the model esterification reaction catalyzed by (1)
the native lipase and the lipase included in the coprecipi-
tates with (2) CeAA, (3) ëÂNH , (4) HDD, (5) CeOH, and
2
(6) PBMA. The lipase content is 0.03 g.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

COPRECIPITATION OF PSEUDOMONAS FLUORESCENS
41
Table 2. Optical rotation of 1-phenylethyl acetate (II) ob- Table 3. The initial lipase activity and the activity of various
tained via acetylation of (1RS)-phenylethanol with vinyl ac- preparations of immobilized lipase in the acetylation of
etate catalyzed by the lipase coprecipitates with various co- (1RS)-phenylethanol with vinyl acetate in tert-butyl methyl
precipitants
ether
Lipase
coprecipitant
Conversion of (1RS)-
phenylethanol, %
Relative activity
of preparations, %
[α]589,* deg
Preparation
Activity*
Native lipase
1.528
3.266
4.628
4.208
1.131
1.200
100
213
303
276
74
–
80.4
76.0
77.2
73.9
70.5
79.2
42.5
46.5
46.0
45.5
48.0
44.8
Lipase on Celite
CeAA
CeNH2
HDD
Lipase and CeAA on Celite
Lipase and HDD on Celite
Lipase on Eupergit C250L
CeOH
PBMA
Lipase and HDD on Eupergit
C250L
78
* In (mmol of 1-phenylethyl acetate) h–1 (mg of lipase)–1.
*
20°C, 1 : 1 acetonitrile–water.
Immobilization of the Lipase on Solid Supports
itself and in the combination with the adsorption on
solid supports.
Despite the possibility of using lipase coprecipitates
with hydrophobic coprecipitants as highly active and
stereospecific catalysts of acetylation in nonaqueous
media, the immobilization of the enzyme on solid car-
riers offers more promise because of the mechanical
stability of such catalysts. In this connection, the immo-
bilization of the lipase on solid supports in the presence
EXPERIMENTAL
A commercially available preparation of the lipase
from Pseudomonas fluorescens (the enzyme content
was 0.25 wt %) with the specific activity for triacetin
–1
–1
of coprecipitants described above is of interest as an hydrolysis of 1200 (µmol of acetic acid) h g was
attempt to obtain highly active and, at the same time, purchased from RÖHM Pharma Polymers (Germany).
mechanically rigid catalysts. We have chosen two prin- Celite 545 was from Serva (Germany). Eupergit
cipally different methods, physical sorption of the C250L, a poly(methyl metacrylate) bearing epoxy
lipase on Celite 545, a zeolite carrier, and a covalent groups, was from Pharma Polymers (Germany). Ethyl
binding of the protein to the Eupergit C250L [a com- acetate (bp 73°ë), dioxane (bp 101°ë), and butyl
mercially available poly(methyl metacrylate) support metacrylate (bp 53°ë/5 mm Hg) (Reakhim, Russia)
that bears epoxy groups] in the presence of those hydro- were purified by distillation. 1-Phenylethanol, vinyl
phobic coprecipitants that displayed high catalytic acetate, tert-butyl methyl ether, and triacetylglycerol
activity (Table 3, Fig. 3).
(triacetin) were from Merck (Germany); N-cetylamine,
Covalent binding of the lipase to Eupergit C250L
led to 74% of the enzymatic activity being retained,
whereas the binding of the lipase treated with HDD to
the same support resulted in 78% of the enzymatic
activity being retained. Therefore, the covalent binding
in the presence of hydrophobic HDD increases, though
not substantially, the esterifying activity of the immobi-
lized enzyme. A much more marked effect was
observed in the case of the lipase sorption on Celite
Yield of (II),%
3
5
4
3
2
0
0
0
0
4
2
1
6
5
5
45: the esterifying activity was 2.1 times more than
that of the native enzyme. Moreover, in the presence of
HDD and CeAA, the esterification rate was further
increased by 1.3 and 1.4 times, respectively. It is obvi-
ous that the presence of hydrophobic coprecipitants
plays an important role in obtaining solid catalysts by
physical sorption of the protein. The physical precipita-
tion of active coprecipitates on the support surface
probably occurs in this case. At the same time, the
coprecipitates cannot be chemically bound to Eupergit
C250L, and, therefore, their activity is almost the same
as that in the absence of coprecipitants. Thus, hydro-
phobic coprecipitation of the lipase can offer both by
10
0
5
10
15
20
25 h
Fig. 3. The kinetic curves of 1-phenylethyl acetate accumu-
lation in the esterification reaction catalyzed by the lipase
immobilized on various supports. The lipase content is
0
.036 mg; the lipase preparations are (1) native lipase; (2)–
(
4) the lipase adsorbed on Celite 545 (2) without coprecipi-
tants and in the presence of (3) CeAA and (4) HDD; the
lipase immobilized on Eupergit C250L (5) without copre-
cipitants and (6) in the presence of HDD.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

4
2
GOROKHOVA et al.
hexadecane-1,2-diol, and cetyl alcohol from Fluka was kept for 24 h at 6°ë. The solid phase was separated
(
Germany); and basic alumina L5/40 from Chemapol by centrifugation (8000 rpm, 20 min), lyophilized, and
(
Czech Republic). AIBN, acetonitrile, acetic anhy- stored at 6–10°ë. The protein content in coprecipitates
dride, triethylamine, hexane, acetone, potassium dihy- was determined from the residual hydrolytic activity of
drogen phosphate (analytical grade), sodium chloride, the enzyme measured in the supernatant taking into
calcium chloride (high-purity grade), and sodium account the lipase content in its commercial prepara-
hydroxide (Reakhim, Russia) were used without addi- tion and the starting activity of the preparation.
tional purification.
Immobilization of the lipase on Celite 545 parti-
(
1)-Phenylethyl acetate (II). Acetic anhydride cles and Eupergit C250L support. The lipase powder
(
0.15 mol), triethylamine (0.15 mol), and ethyl acetate (80 mg) was dissolved at 0°ë in buffer A (10 ml), and
25 ml) were added to 0.05 mol of RS-(I). The reaction separated from insoluble impurities by filtration. Celite
(
mixture was refluxed for 4 h and washed three times 545 (1 g) was added, the mixture was incubated for 72 h
with 1 M äç êé (pH 4.5), and the organic phase was at 6°ë, and then the water was removed. The resulting
2
4
distilled to give RS-(II), bp 89°C/5 mm Hg [15].
enzyme preparation was stored at 6–10°ë.
Preparation of the immobilized enzyme was simi-
larly obtained from 100 mg of the lipase powder solu-
tion in 10 ml of buffer A and 1 g of Eupergit C250L by
incubation for 24 h at 6°ë. The supernatant was
decanted, and its hydrolytic activity was measured to
calculate the amount of lipase covalently bound to the
support. The preparation of immobilized enzyme was
dried on a porous glass filter by blowing air and stored
at 6–10°ë.
RS-(I), vinyl acetate, and (II) were separated in a
model experiment on a LiChroCART RP 18 (5 µm)
column (240 × 4 mm, Merck, Germany) on a Beckman
System Gold instrument (United States). Optical
absorption was measured at 260 nm. Optimal condi-
tions for HPLC were as follows: flow rate of
0
.8 ml/min, 1 : 1 acetonitrile–water as an eluent, ten-
fold dilution of the mixture under study with the eluent,
and an applied sample volume of 20 µl at (RS-(I) and
vinyl acetate contents of 0.5 and 1.5 µmol, respectively.
The preparations of the lipase suspension with
Under these conditions, the retention times for RS-(I), coprecipitants obtained as described above were immo-
vinyl acetate, and RS-(II) were 5.9, 6.2, and 13.5 min, bilized similarly.
respectively. Mixtures of (I) and (II) containing 10, 20,
The hydrolytic activity of the dissolved and
3
1.4, and 40% of (II) according to GC were used as
immobilized lipase was determined in the reaction of
triacetin hydrolysis. A solution (50 µl) containing the
enzyme (2.5 µg) or the dry coprecipitate (0.5 mg) was
control samples. Contents of (II) determined by
rpHPLC were 9.7, 19.4, 30.3, and 41.7%, respectively.
N-Cetylacetamide. Acetic anhydride (50 mmol) added to a solution (5 ml) of the substrate containing
was added dropwise to a stirred solution of ëÂNH2 0.1 M triacetin, 0.05 M sodium chloride, and 0.05 M
25 mmol) in ethyl acetate (200 ml), and the mixture calcium chloride in the cell of a Radiometer Copen-
(
was stirred for an additional 2 h. The reaction mixture hagen TTT 60 titrator. Acetic acid released in the enzy-
was washed three times with water and evaporated in a mic reaction was titrated with 0.01 M NaOH solution to
vacuum, and the resulting CeAA was recrystallized pH 7.0 for 10–15 min. The hydrolytic activity of the
from hexane and dried in a desiccator, mp 85°ë [16].
Poly(butyl metacrylate). To remove stabilizing
hydroquinone, monomeric butyl metacrylate was dis-
enzyme was calculated from the titration results in
–1
–1
(µmol of acetic acid) h (mg of the enzyme) .
The activity of the lipase and its immobilized
solved in hexane, passed through the column with alu- preparations in the esterification reaction was deter-
mina, and distilled. It was polymerized in bulk at 80°ë mined from the initial rate of the of RS-(I) acetylation
after preliminary nitrogen bubbling with AIBN (3% of with vinyl acetate [7]. RS-(I) (5 mmol) and vinyl acetate
polymer mass) as an initiator. The resulting polymer (15 mmol) were added to TBME (10 ml). Then the
was dissolved in dioxane, separated from the monomer lipase powder (14 mg), the lipase coprecipitate (2 mg),
by reprecipitation with ethanol, and dried in a desicca- or the solid carrier with the immobilized lipase
tor with a yield of 60%. The molecular mass of PBMA (300 mg) was added, and the mixture was shaken at
determined viscosimetrically by the standard procedure 37°ë. The Aliquots (0.5 ml) were sampled at defined
[
17] was 20 000.
intervals (Figs. 2, 3), the solid catalyst particles were
separated by centrifugation (10 000 rpm, 5 min), and
the supernatant was analyzed as described above. The
initial rate of the enzymic reaction was calculated from
the accumulation rate of the reaction product (II) in
Lipase inclusion into the hydrophobic coprecipi-
tates with CeOH, ëÂNH , CeAA, and PBMA was car-
2
ried out as described for its coprecipitates with HDD
according to the procedure reported earlier [12]. The
lipase (0.2 g) was dissolved in 10 ml of 0.01 M phos-
phate buffer, pH 7.5 (buffer A) at 0°ë and separated
–
1
–1
µmol h (mg of the enzyme) .
The determination of the acetylation enantiose-
from insoluble impurities by filtration. A solution of lectivity under the catalysis by lipase preparations.
HDD (20 mg) in warm acetone (0.7 ml) was added To isolate (II), obtained via catalysis by various lipase
dropwise to the lipase solution under stirring. Acetone coprecipitates, the reaction mixture (10 ml) was con-
was removed in a vacuum, and the aqueous solution centrated in a vacuum to the volume of 2.5 ml, solid
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

COPRECIPITATION OF PSEUDOMONAS FLUORESCENS
43
particles of catalyst were separated by centrifugation,
the supernatant was diluted fivefold with the eluent, and
the aliquot was chromatographed as described above.
The fraction whose retention time corresponded to that
of model (II) was isolated by HPLC. The optical rota-
tion of the product was measured on a Digital Polarim-
eter DIP-360 (Jasco, Japan) at 589 nm (Na D-line) in
7. Laumen, K., Breitgoff, D., and Schneider, M.P., J. Chem.
Soc., Chem. Commun., 1988, vol. 18, pp. 1459–1461.
8. Norin, M., Boutelje, J., Holmberg, E., and Hult, K.,
Appl. Microbiol. Biotechnol., 1988, vol. 28, pp. 527–
530.
9
. Bosley, J.A. and Clayton, J.C., Biotechnol. Bioeng.,
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1
1
: 1 acetonitrile–water at 20°ë.
1
0. Ivanov, A.E. and Schneider, M.P., J. Mol. Catal., B,
Enzymatic, 1997, vol. 3, pp. 303–309.
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