Fine-tuning the second generation sol-gel lipase immobilization with ternary alkoxysilane precursor systems

Article abstract of DOI:10.1016/j.procbio.2010.07.021

The sol-gel immobilization of Celite-supported lipase from Pseudomonas fluorencens (Lipase AK) was systematically studied using ternary silane precursor systems consisting of alkyltriethoxysilane (alkylTEOS), phenyltriethoxysilane (PhTEOS) and tetraethoxysilane (TEOS). The parameters investigated were the surface coverage at various enzyme-Celite ratios (between 1:1 and 1:10) and the effect of molar ratio of alkylTEOS (alkyl = propyl, hexyl, octyl, 1H,1H,2H,2H-perfluorooctyl, decyl, dodecyl, octadecyl), PhTEOS and TEOS (seven series of alkylTEOS:PhTEOS:TEOS from 0.1:0.9:1 to 0.9:0.1:1 in 0.1 steps) on the catalytic properties of the sol-gel biocatalysts. For comparison, the corresponding binary systems (1:1 molar ratios of alkylTEOS:TEOS and PhTEOS:TEOS) were also studied. The ternary and binary sol-gel lipase preparations were evaluated by their catalytic behavior in enantiomer selective acetylation of racemic 1-phenylethanol and 2-heptanol. For each alkylTEOS precursor, one or more alkylTEOS/PhTEOS/TEOS ternary systems were superior biocatalysts compared to the corresponding alkylTEOS/TEOS preparations. The best overall results were achieved with the medium-chain octylTEOS and perfluorooctylTEOS-containing ternary systems.

Full text of DOI:10.1016/j.procbio.2010.07.021

Process Biochemistry 46 (2011) 52–58
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Fine-tuning the second generation sol–gel lipase immobilization with ternary
alkoxysilane precursor systems
a
a
a,b,c
a
d
Anna Tomin , Diana Weiser , Gabriella Hellner
, Zsófia Bata , Livia Corici ,
d
e
a,∗
Francisc Péter , Béla Koczka , László Poppe
Department of Organic Chemistry and Technology, and Research Group for Alkaloid Chemistry of the Hungarian Academy of Sciences,
Budapest University of Technology and Economics, H-1111 Budapest, M u˝ egyetem rkp. 3, Hungary
Department of Microbiology and Biotechnology, Corvinus University of Budapest, H-1118 Budapest, Somlói út 14-16, Hungary
Bunge Europe Innovation Centre, H-1097 Budapest, Illatos út 38, DÜP II, Building G, 3rd Floor, Hungary
a
b
c
d
Department of Applied Chemistry and Engineering of Organic and Natural Compounds, University Politehnica of Timisoara,
Faculty of Industrial Chemistry and Environmental Engineering, Str. C. Telbisz 1, RO-300001 Timisoara, Romania
e
Department of General and Inorganic Chemistry, Budapest University of Technology and Economics, H-1111 Budapest, Gellért tér 4, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The sol–gel immobilization of Celite-supported lipase from Pseudomonas fluorencens (Lipase AK) was
systematically studied using ternary silane precursor systems consisting of alkyltriethoxysilane (alkyl-
TEOS), phenyltriethoxysilane (PhTEOS) and tetraethoxysilane (TEOS). The parameters investigated were
the surface coverage at various enzyme-Celite ratios (between 1:1 and 1:10) and the effect of molar
ratio of alkylTEOS (alkyl = propyl, hexyl, octyl, 1H,1H,2H,2H-perfluorooctyl, decyl, dodecyl, octadecyl),
PhTEOS and TEOS (seven series of alkylTEOS:PhTEOS:TEOS from 0.1:0.9:1 to 0.9:0.1:1 in 0.1 steps) on the
catalytic properties of the sol–gel biocatalysts. For comparison, the corresponding binary systems (1:1
molar ratios of alkylTEOS:TEOS and PhTEOS:TEOS) were also studied. The ternary and binary sol–gel lipase
preparations were evaluated by their catalytic behavior in enantiomer selective acetylation of racemic
1-phenylethanol and 2-heptanol. For each alkylTEOS precursor, one or more alkylTEOS/PhTEOS/TEOS
ternary systems were superior biocatalysts compared to the corresponding alkylTEOS/TEOS preparations.
The best overall results were achieved with the medium-chain octylTEOS and perfluorooctylTEOS-
containing ternary systems.
Received 28 April 2010
Received in revised form 15 June 2010
Accepted 9 July 2010
Dedicated to Professor Károly Lempert on
his 85th birthday.
Keywords:
Sol–gel
Ternary silane-precursor system
Immobilization
Lipase
Biocatalysis
Kinetic resolution
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
ammoniolysis [1,7,8]. Theesterification, interesterification reaction
or ester hydrolysis usually proceed with high regio- and/or enan-
tioselectivity, making lipases an important group of biocatalysts in
organic chemistry.
The development of biotechnology brings along the immobi-
lization of biomolecules or microorganisms. For further indus-
trial applications, immobilization of enzymes has been an
active research topic in enzyme technology to enhance their
activity, thermal and operational stability, and reusability
[9–11].
Among many available immobilization methods, including
adsorption, covalent attachment to solid supports and entrap-
ment within polymers [9,10], entrapment of enzymes in inor-
ganic/organic hybrid polymer matrices has received a lot of
attention in recent years and has provided new possibilities in the
field of material science [12,13]. Sol–gel encapsulation has proven
to be a particularly easy and effective way to immobilize puri-
fied enzymes, whole cells, antibodies and other proteins [14–16].
Sol–gel immobilization of lipases can enhance their thermostability
Lipases (EC 3.1.1.3) are versatile hydrolytic enzymes that can
be obtained from animals and plants as well as from natural
and recombinant microorganisms with good yields [1,2]. Lipases
perform essential roles in the digestion, transport and process-
ing of lipids (e.g. triglycerides, fats, oils) in most, if not all, living
organisms. Microbial lipases serve important everyday life roles
as ancient as yogurt and cheese fermentation. However, lipases
are also being exploited as cheap and versatile biocatalysts in
more modern applications [3–5]. For instance, lipases are used in
applications such as baking and laundry detergents and even as
biocatalysts in alternative energy strategies to convert vegetable
oil into biofuel [6]. Lipases are flexible biocatalysts which can cat-
alyze a wide range of enantio- and regioselective reactions such
as hydrolysis, esterifications, transesterifications, aminolysis and
∗ Corresponding author. Tel.: +36 1 4632229; fax: +36 1 463 3697.
E-mail address: poppe@mail.bme.hu (L. Poppe).
[
19,17], long-term operational stability and storage life [24,25].
1
359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.07.021

A. Tomin et al. / Process Biochemistry 46 (2011) 52–58
2.3. Scanning electron microscopy
53
A typical sol–gel immobilization process involves acid- or
base-catalyzed hydrolysis, then polycondensation of alkoxysilane
The surface morphology of the samples was investigated with a JEOL JSM-
500LV scanning electron microscope. The samples of free Lipase AK, Lipase AK
on solid support and supported Lipase AK encapsulated in sol–gel matrices were
coated with gold prior to analysis. Electron beam energy of 25 kV was used for the
investigations.
precursor [Si(OR) ] in the presence of additives to form a matrix
4
5
in which the enzyme is encapsulated. Using organically modi-
ꢀ
fied silanes of the type R –Si(OR) , activity enhancement of the
3
immobilized lipases could be observed with increasing amount
ꢀ
and alkyl chain (R ) length of the hydrophobic silanes [18–26]. The
2
.4. Activity and enantiomer selectivity tests of the Lipase AK preparations
optimal molar ratios of the trialkoxy- and tetraalkoxysilane pre-
ꢀ
cursors [R –Si(OR)3 and Si(OR) ] were also investigated [18,24,26].
4
The free or sol–gel immobilized Lipase AK (50 mg) was added to the solution of
The presence of additives like polyethylene glycol, polyhydroxy
compounds or proteins [25] can significantly enhance the catalytic
activity of these enzymes. It was demonstrated that the presence of
a small amount of isopropyl alcohol (IPA) during immobilization is
beneficial [27,28]. The characterization of the sol–gel biocatalysts
is also well documented [29].
racemic test alcohol (1-phenylethanol, rac-1a, 49 mg, 0.4 mmol; or 2-heptanol, rac-
1
b, 46 mg, 0.4 mmol) and vinyl acetate (100 ␮l) in hexane – THF 2:1 (1 ml), and the
◦
resulting mixture was shaken at 30 C in a sealed glass vial at 1000 cycles per minute.
For GC analyses, samples were taken directly from the reaction mixture (sample size:
10 ␮l, diluted with CH2Cl2 to 100 ␮l) at 2, 4 and 8 h. The esters were analyzed on
an Acme 6100 instrument (Young Lin Instrument Co., South Korea) equipped with
flame ionization detector and Hydrodex ␤-6TBDM [30 m × 0.25 mm × 0.25 ␮m film
of heptakis-(2,3-di-O-methyl-6-O-t-butyldimethylsilyl)-␤-cyclodextrin (Macherey
The sol–gel immobilized lipases can be supported on inert mate-
rials such as Celite to improve the diffusion of the substrates or
products to and from the enzyme and thus improve the reaction
rate [21,26,30–33]. Mesoporous silica materials can be also effec-
tive supports during immobilization [34].
&
Nagel)] column (oven temperature, injector and detector temperatures were
◦
◦
◦
−1
130 C, 250 C and 250 C, respectively; carrier gas:H2 at a flow of 1.8 ml min ;
split ratio:1:50). Data on conversion, enantiomeric composition of the products
[(R)-2a,b and (S)-1a,b] and the calculated properties of the enzyme are presented in
Tables 1 and 2 and Fig. 3.
−
1
−1
ꢀ
The effective specific activity of the biocatalyst [UB (␮mol × min × g )] could
Although it is well known that the nature of the R substituent in
be determined in the test reaction from the amount of the racemic alcohol [nrac
the trialkoxysilane precursors may significantly influence the prop-
erties of the sol–gel lipases [18–26], only the binary sol–gel systems
(
␮mol)], the conversion [c], the reaction time [t (min)] and the mass of the free
biocatalysts (LAK) or the free biocatalyst subjected to the sol–gel immobilized (imm-
LAK) [mB (g)].
ꢀ
of trialkoxy- and tetraalkoxysilane precursors [R –Si(OR)3 and
Si(OR) ] for lipase immobilization have been studied [18,24,26].
nracc
4
UB =
According to our best knowledge, ternary sol–gel systems of
a tetraalkoxysilane and two different trialkoxysilane precursors
tmB
As the effective specific activities in the present work were calculated from a single-
point experiment, the term “activity” must be considered only for comparative
evaluation of the catalytic efficiencies, not for the real kinetic behavior of the bio-
catalyst.
The activity yield [YA (%)] can be calculated from the effective specific activity of
the immobilized biocatalyst (UB,imm-LAK) compared to the effective specific activity
of the free Lipase AK (UB,LAK). UB,imm-LAK can be determined in the test reaction from
the amount of the racemic alcohol [nrac (␮mol)], the conversion [c], the reaction time
1
2
[
R –Si(OR) , R –Si(OR) and Si(OR) ] have been studied only for
3 3 4
cutinase (EC 3.1.1.74) [33]. Therefore, our goal was to perform a sys-
tematic study of the sol–gel immobilization of a Celite-supported
lipase using ternary silane precursor systems of various alkyl-
triethoxysilanes (alkylTEOS), phenyltriethoxysilane (PhTEOS) and
tetraethoxysilane (TEOS).
[
t (min)] and the mass of the free lipase subjected to the sol–gel immobilization [mB
(
g)].
2. Materials and methods
1
00UB,imm-LAK
(nracc)/(tmB)
UB,LAK
YA =
= 100
2.1. Materials
UB,LAK
The enantiomer selectivity in a kinetic resolution can be calculated from two of the
following three data: conversion [c], enantiomeric excess of the remaining alcohol
Lipase AK (lipase from Pseudomonas fluorescens, Cat. No. 534730), 2-propanol
(
IPA), vinyl acetate and sodium fluoride (NaF) were products of Aldrich. 1-
®
[ee
(S)−1
(R)−2
], enantiomeric excess of the forming ester [ee ] [35,36].
Phenylethanol, 2-heptanol, polyethylenglycol 1000 (PEG), Celite 545 (Cat. No.
2140), tetraethoxysilane (TEOS), n-propyltriethoxysilane (PrTEOS) and phenyl-
triethoxysilane (PhTEOS) were obtained from Fluka. n-Hexyltriethoxysilane
HexTEOS), n-octyltriethoxysilane (OctTEOS), n-decyltriethoxysilane (DecTEOS),
n-dodecyltriethoxysilane (DodTEOS), n-octadecyltriethoxysilane (OctdTEOS), and
H,1H,2H,2H-perfluorooctyltriethoxysilane (PFOctTEOS) were obtained from Alfa
Aesar.
2
3. Results and discussion
(
The beneficial properties of the sol–gel immobilized lipases may
1
ꢀ
be significantly influenced by the nature of the R substituent in
binary sol–gel systems of trialkoxy- and tetraalkoxysilane precur-
ꢀ
2
.2. Sol–gel immobilization of Lipase AK deposited on Celite® 545 using binary or
sors [R –Si(OR)3 and Si(OR) ] [18–26] but ternary sol–gel systems
4
ternary silane precursor systems
of a tetraalkoxysilane and two different trialkoxysilane precur-
1
2
sors [R –Si(OR) , R –Si(OR) and Si(OR) ] had not been studied for
3
3
4
A two-step procedure was applied for sol–gel entrapment of Lipase AK on Celite
lipase immobilization.
5
3
45.
Step 1: The Lipase AK powder (50 mg for the standard procedure; 50, 125, 250,
75 or 500 mg for the enzyme loading tests) was added to Tris–HCl buffer (0.1 M,
3.1. Selection of the lipase, support and silane precursors
◦
pH 7.5, 780 ␮l) at 4 C with stirring for 10 min followed by addition of Celite 545
(
1
500 mg). Acetone (10 ml) was added to the well stirred Celite-enzyme mixture at
0 ml min rate at −18 C. The resulting solid was filtered off and left in air at room
In most cases, alkyltrimethoxysilanes (alkylTMOS’s) were pre-
ferred for encapsulation of lipases. However, it was indicated
that there is no significant difference in properties of encapsu-
lated lipase biocatalysts prepared from alkylTMOS or alkylTEOS
silane precursors [31]. Because alkylTEOS’s are cheaper and the
gelation time is longer and more controllable than with alkylT-
MOS’s, the use of R –Si(OEt)3 and Si(OEt)4 as silane precursors
is preferable. The second generation sol–gel immobilized lipases
are supported on inert materials to reduce diffusion limitations
−
1
◦
temperature for 12 h for drying. The Celite-enzyme preparations were then used for
sol–gel immobilization.
Step 2: Tris–HCl buffer (0.1 M, pH 7.5, 390 ␮l), PEG solution (4%, w/v, 200 ␮l),
NaF solution (1 M, 100 ␮l) and IPA (200 ␮l) were mixed in a 20 ml glass vial and
the resulting solution was shaken at 1000 cycles per minute at room tempera-
ture for 10 min. During the continuous shaking, the corresponding silane precursors
alkylTEOS–TEOS (1.5 mmol alkylTEOS and 1.5 mmol TEOS) or the mixture of alkyl-
TEOS:PhTEOS:TEOS (3 mmol; the alkylTEOS:PhTEOS:TEOS molar ratio varied from
ꢀ
0
.1:0.9:1 to 0.9:0.1:1 in 0.1 steps) and Celite-enzyme (250 mg) were added to the
vial resulting in a sol suspension. To complete the polymerization, the mixture was
shaken for 12 h at room temperature. The formed solid was washed with IPA (7 ml),
distilled water (5 ml), IPA (5 ml) and n-hexane (5 ml). The resulting white powder
was dried in a vacuum exicator for 5 h (until 0.4 mm Hg final level of vacuum). The
sol–gel Lipase AK preparations were stored at room temperature.
[
21,26,30–33]. The lipase from Pseudomonas fluorescens (Lipase
AK) proved to be a good lipase model for studies of factors influ-
encing the sol–gel immobilization of lipases [22,31]. Because the
esterification activity of immobilized lipases can be enhanced by

5
4
A. Tomin et al. / Process Biochemistry 46 (2011) 52–58
Table 1
Characterization of Celite-supported Lipase AK immobilized in binary sol–gel systems of alkylTEOS/TEOS silane precursors. Effective specific activity (UB), activity yield (YA)
and enantiomer selectivity (E) of the free and immobilized forms of Lipase AK are shown in Panel A (with 1-phenylethanol rac-1a) and Panel B (with 2-heptanol rac-1b).
No.
Silane precursorsa
Substrate
Time (h)
c (%)
Eb
UBc (U g−1
)
YA (%)
Panel A
1
2
3
4
5
6
7
8
- (Lipase AK)
PrTEOS:TEOS
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
0.25
8
2
4
2
8
8
8
2
28
24
28
25
24
33
27
29
39
ꢁ200
ꢁ200
>200
ꢁ200
>200
>200
ꢁ200
>200
ꢁ200
154.2
4.1
19.0
8.4
16.8
5.7
4.6
100
60
181
77
156
54
44
HexTEOS:TEOS
OctTEOS:TEOS
PFOctTEOS:TEOS
DecTEOS:TEOS
DodTEOS:TEOS
OctdTEOS:TEOS
PhTEOS:TEOS
5.0
26.8
44
242
9
Panel B
10
11
12
13
14
15
16
17
18
- (Lipase AK)
PrTEOS:TEOS
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
1
8
8
8
8
8
8
8
8
28
0.3
17
8
22
8
4
5
30
13.8
38.4
0.1
2.9
1.3
3.7
1.3
0.6
0.9
5.2
100
7
109
49
139
51
25
1.2
12.6
10.8
12.2
11.0
10.6
10.0
14.7
HexTEOS:TEOS
OctTEOS:TEOS
PFOctTEOS:TEOS
DecTEOS:TEOS
DodTEOS:TEOS
OctdTEOS:TEOS
PhTEOS:TEOS
32
187
a
Lipase AK was preadsorbed on Celite 545 at 1:10 enzyme:support ratio and entrapped with alkylTEOS:TEOS silane precursors at 1:1 molar ratio.
The enantiomer selectivity (E) was calculated from c and ee(S)−1/ee(R)−2 [35,36]. Due to sensitivity to experimental errors, E values calculated in the 100–200 range are
b
reported as >100, values in the 200–500 range are reported as >200 and values calculated above 500 are given as ꢁ200.
c
In entries 1 and 10 UB refers to the free Lipase AK (UB,LAK) and in entries 2–9 and 11–18 UB refers to the amount of free Lipase AK subjected to immobilization (UB,imm-LAK).
forming the entrapping-gel on the surface of Celite [19,26], fine-
tuning the sol–gel immobilization process of Lipase AK deposited
on Celite 545 was selected as the subject for this study. Thus,
the properties of Celite-supported Lipase AK immobilized with
ternary silane precursor systems of various alkyltriethoxysilanes
was applied. Thus, to select the ideal enzyme distribution on
the solid support in the first step, various amounts of Lipase
AK were adsorbed on Celite 545 as solid support by precip-
itation from aqueous solution with the slow addition of cold
acetone.
(
(
alkylTEOS), phenyltriethoxysilane (PhTEOS) and tetraethoxysilane
TEOS) were investigated in this work.
Celite, also known as diatomite or kieselgur, is a naturally
occurring, inexpensive fossilic diatomaceous earth. It consists of
fossilized remains of diatoms, a type of hard-shelled algae, ranging
in size from ca. 2 to 200 ␮m [37], that are composed of a cell wall
comprising silica [38]. This siliceous wall can be highly patterned
with a variety of pores, ribs, minute spines, marginal ridges and
elevations.
3
.2. Adsorption of Lipase AK on Celite 545
A two-step immobilization protocol involving pre-adsorption
of the enzyme on solid support followed by sol–gel entrapping
Table 2
Characterization of the best performing Celite-supported ternary sol–gel Lipase AK biocatalysts prepared from alkylTEOS/PhTEOS/TEOS silane precursors. Effective specific
activity (UB), activity yield (YA) and enantiomer selectivity (E) of the free and immobilized forms of Lipase AK are shown in Panel A (with 1-phenylethanol rac-1a) and Panel
B (with 2-heptanol rac-1b).
No.
Triethoxy silane precursorsa
Substrate
AlkylTEOS amountb (%)
Time (h)
c (%)
Ec
UBd (U g−1
)
YA (%)
Panel A
1
2
3
4
5
6
7
8
- (Lipase AK)
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
rac-1a
–
60
60
70
90
50
30
60
0
0.25
8
4
4
2
8
2
4
2
28
25
35
34
24
27
20
30
39
ꢁ200
ꢁ200
>200
ꢁ200
ꢁ200
ꢁ200
ꢁ200
ꢁ200
ꢁ200
154.2
4.3
12.2
11.8
16.7
4.6
13.6
10.2
26.8
100
52
116
109
181
18
53
99
242
PrTEOS:PhTEOS
HexTEOS:PhTEOS
OctTEOS:PhTEOS
PFOctTEOS:PhTEOS
DecTEOS:PhTEOS
DodTEOS:PhTEOS
OctdTEOS:PhTEOS
PhTEOS
9
Panel B
10
11
12
13
14
15
16
17
18
- (Lipase AK)
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
rac-1b
–
60
60
70
90
50
30
60
0
1
8
8
8
8
8
8
8
8
28
4.7
6
19
27
2.6
21
16
30
13.8
38.4
0.8
1.0
3.2
4.7
0.1
3.6
2.8
5.2
100
40
35
64
205
32
128
108
187
PrTEOS:PhTEOS
HexTEOS:PhTEOS
OctTEOS:PhTEOS
PFOctTEOS:PhTEOS
DecTEOS:PhTEOS
DodTEOS:PhTEOS
OctdTEOS:PhTEOS
PhTEOS
11.6
10.2
12.3
12.3
8.3
13.5
15.6
14.7
a
Lipase AK was preadsorbed on Celite 545 (1:10 enzyme:support ratio) and entrapped in a sol–gel from alkylTEOS:PhTEOS:TEOS silane precursors at x:(100 − x):100 molar
ratio.
b
Amount (x) of alkylTEOS in the triethoxysilane mixture.
The enantiomer selectivity (E) was calculated from c and ee(S)−1/ee(R)−2 [35,36]. Due to sensitivity to experimental errors, E values calculated in the 100–200 range are
c
reported as >100, values in the 200–500 range are reported as >200 and values calculated above 500 are given as ꢁ200.
d
In entries 1 and 10 UB refers to the free Lipase AK (UB,LAK) and in entries 2–9 and 11–18 UB refers to the amount of free Lipase AK subjected to immobilization (UB,imm-LAK).

A. Tomin et al. / Process Biochemistry 46 (2011) 52–58
55
Fig. 1. SEM images of Celite (A), free Lipase AK (B), Celite-supported Lipase AK immobilized in octylTEOS/PhTEOS/TEOS 0.7/0.3/1 sol–gel (C) and various amounts of Lipase
AK precipitated onto Celite [Celite/Lipase AK ratio: 10:1 (D), 4:1 (E), 10:5 (F), 4:3 (G) and 1:1 (H)].
To study the surface coverage of Celite, supported lipase AK
preparations of different enzyme-Celite ratios (1:10, 1:4, 1:2, 3:4
and 1:1) were made. The free Celite (Fig. 1(A)), free Lipase AK
(Fig. 1(A)) are the best forms to investigate the coverage of the solid
support (Fig. 1(C)–(H)).
The SEM pictures indicated that at increased amounts of the
precipitated enzyme on Celite more and more monoclinic or
orthorhombic particles were formed on the surface of the support
(Fig. 1(D)–(H)). It is known that lipases from different Pseudomonas
strains crystallize in similar crystal forms; for example the lipase
from P. cepacia (PDB code: 1YS1) forms monoclinic crystals [39],
while the lipase from P. aeruginosa (PDB code: 1EX9) has orthorom-
bic crystal structure [40]. Because the lipase from P. fluorescens
(Uniprot code A9YY76) exhibits high amino acid sequence homol-
ogy with lipase of P. cepacia (37% identity, 53% homology) and P.
aeruginosa (45% identity, 60% homology), it can be supposed that
(
Fig. 1(B)), a sol–gel entrapped Lipase AK on Celite (Fig. 1(C)) and
Celite preparations covered with various amounts of Lipase AK
(
(
Fig. 1(D)–(H)) were inspected by scanning electron microscopy
SEM).
The SEM investigations indicated that the commercial Lipase AK
consists of relatively large spherical aggregates (4–40 ␮m diameter,
Fig. 1(B)). This size distribution indicates that substantial diffusion
limitation may occur when the free form of Lipase AK is used for
catalysis in organic media. Visual inspection of the SEM images
of the free Celite revealed that the disc-shaped particles in Celite

5
6
A. Tomin et al. / Process Biochemistry 46 (2011) 52–58
andactivityofthe freeLipase AKunderidentical reaction conditions
and conversion ranges were the references in all the cases.
ꢀ
It had already been indicated that mixtures of R -Si(OEt)3
and Si(OEt)4 at 1:1 molar ratio provided ideal matrices for
the sol–gel process [18]. Accordingly, the binary systems
[
R–Si(OEt) :TEOS = 1:1] of eight different triethoxysilanes (PrTEOS,
3
HexTEOS, OctTEOS, PFOctTEOS, DecTEOS, DodTEOS, OctdTEOS,
PhTEOS) were investigated first in the acylation reaction of 1-
phenylethanol (rac-1a, Panel A of Table 1) and 2-heptanol (rac-1b,
Panel B of Table 1). In the cases of HexTEOS, PFOctTEOS and PhTEOS,
Fig. 2. Kinetic resolution of racemic secondary alcohols rac-1a,b as test reaction for
characterization of the free and immobilized Lipase AK biocatalysts.
the activity yield (Y ) for the binary sol–gel entrapped Lipase AK
A
exceeded 100% with both test alcohols (Table 1). The best enan-
tiomer selectivities (E) were achieved with the binary systems
of PrTEOS, OctTEOS, DodTEOS and PhTEOS for rac-1a (Panel A
of Table 1) and with the binary sol–gel biocatalysts containing
HexTEOS, OcTEOS, PFOctTEOS and PhTEOS for rac-1b (Panel B of
Table 1).
Lipase AK of P. fluorescens origin forms similar crystals (typically
of 1–5 ␮m size, Fig. 1(F)–(H)) as lipases from P. aeruginosa or P.
cepacia. It is then understandable that the effective specific activ-
ity of the enzyme decreases at higher enzyme loading due to the
increasing diffusion limitation within these relatively large crys-
tals. According to the morphology studies, the 1:10 enzyme-Celite
ratio provided thin and uniform coating on the surface of Celite
From the data with the R–Si(OEt) :TEOS = 1:1 systems, it was
3
obvious that among the binary systems the PhTEOS:TEOS = 1:1
composition resulted in optimal properties regarding both activity
and selectivity.
Next, by keeping the beneficial PhTEOS as one of the three
silane precursors, we tried fine-tuning of the sol–gel system by
using ternary systems of alkylTEOS:PhTEOS:TEOS silane precur-
sors in various ratios. Thus, the alkylTEOS:PhTEOS molar ratio was
varied from 0.1 to 0.9 in 0.1 steps while keeping the trialkoxysi-
lane (alkylTEOS:PhTEOS):tetraalkoxisilane (TEOS) molar ratio at
(
Fig. 1(D)) and was selected for the further investigations. The
SEM image of one of the best performing ternary sol–gel immo-
bilized preparations (Fig. 1(C); Celite:Lipase AK 10:1, entrapped
in octylTEOS/PhTEOS/TEOS 0.7/0.3/1 sol–gel) indicated that sur-
face coating of Celite remained uniform and thin after the sol–gel
entrapping step as well.
3.3. Effect of the composition of alkylTEOS–PhTEOS–TEOS silane
1
:1. The properties of the resulting series of ternary sol–gel biocat-
precursors on the properties of sol–gel Lipase AK supported on
Celite 545
alysts were characterized by testing their catalytic behavior in the
kinetic resolution of 1-phenylethanol rac-1a (Fig. 3). The effective
specific activity (UB), activity yield (Y ) and enantiomer selectiv-
ity (E) values of the enantiomer selective acetylation of racemic
A
The effect of the silane precursor composition on enantiomer
selectivity and catalytic ability were investigated in the kinetic res-
olution of racemic secondary alcohols (Fig. 2). All the binary and
ternary sol–gel preparations were tested with the selective acyla-
tion of 1-phenylethanol (rac-1a), in hexane-THF using vinyl acetate
as acyl donor (Fig. 3, Panels A in Tables 1 and 2). Acylation of racemic
1
-phenylethanol rac-1a, calculated at the initial part of the reac-
tion (at 4 h), were compared for the ternary sol–gel Lipase AK
biocatalysts, the PhTEOS:TEOS binary sol–gel immobilized Lipase
AK and free Lipase AK. Comparison of the ternary systems to the
best binary biocatalyst (PhTEOS:TEOS = 1:1) at 4 h indicated that
properties of numerous ternary compositions were superior to the
PhTEOS:TEOS binary system in all the aspects (Fig. 3). In general,
the UB and YA values, related to the productivity of the biocatalysts
with racemic 1-phenylethanol rac-1a, were the best for ternary
compositions prepared from medium chain alkylsilane precursors
2
-heptanol (rac-1b), exhibiting moderate enantiomer selectivity
with free Lipase AK, was also investigated with the binary (Panel
B of Table 1) and selected ternary sol–gel preparations (Panel B of
Table 2). To evaluate the efficiency of the immobilization and bio-
catalysts, the specific activities (UB), and activity yields (Y ) and
A
enantiomer selectivities (E) with substrate rac-1a were compared
at ∼30% conversion in all the cases. The enantiomer selectivity (E)
(HexTEOS, OctTEOS, PFOctTEOS), while enantiomer selectivities (E)
Fig. 3. Characterization of Celite-supported ternary sol–gel Lipase AK biocatalysts prepared from alkylTEOS/PhTEOS/TEOS silane precursors. Effective specific activity, UB (A);
activity yield, YA (B) and enantiomer selectivity, E (C) of the free and immobilized forms of Lipase AK are calculated at 4 h reaction time for enantiomer selective acetylation
of racemic 1-phenylethanol rac-1a. The octylTEOS series and representatives of the other alkylTEOS systems included in Table 2 are shown in black.

A. Tomin et al. / Process Biochemistry 46 (2011) 52–58
57
were sufficient for almost all the longer alkylTEOS precursors (Hex-
TEOS, OctTEOS, PFOctTEOS, DecTEOS, DodTEOS, OctdTEOS). Among
all the ternary systems, the perfluorinated chain containing PFOct-
TEOS series exhibited the best overall performance. Taking the price
of PFOctTEOS also into account, however, the OctTEOS series pro-
vided the best performance/price result in the kinetic resolution of
positions were optimal for the two different substrates. Although
the medium-chain octylTEOS- and perfluorooctylTEOS-containing
ternary systems performed the best in general, this study indicated
that individual fine-tuning might develop the best biocatalyst for
each individual substrate.
1
-phenylethanol rac-1a.
Next, the OctTEOS series and a well performing member of
Acknowledgements
each other alkylTEOS ternary series (marked in black in Fig. 3)
were investigated in the kinetic resolution of 2-heptanol rac-1b,
a moderate substrate of Lipase AK. Comparison of the data from
the kinetic resolutions of the two different secondary alcohols,
This research work was supported by the Hungarian National
Office for Research and Technology (NKFP-07-A2 FLOWREAC) and
by the Hungarian National Science Foundation (OTKA T-048854).
L.C. thanks the fellowship supported by the Ministry of Labour,
Family and Social Protection, Romania (POSDRU 6/1.5/S/13, 2008),
co-financed by the European Social Fund - Investing in People.
1
-phenylethanol rac-1a and 2-heptanol rac-1b with the binary
(
Table 1) and the selected ternary (Table 2) sol–gel systems revealed
that different silane precursor compositions resulted in the best
activities and selectivities for the two substrates rac-1a,b (Panels A
for rac-1a and Panels B for rac-1b in Tables 1 and 2, respectively).
In the case of the kinetic resolution of 1-phenylethanol rac-1a
at around 30% conversion (Panels A in Tables 1 and 2), the addi-
tion of PhTEOS to longer chain alkylTEOS (OctTEOS, PFOctTEOS,
DecTEOS, DodTEOS, OctdTEOS) resulted in ternary sol–gel systems
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