Preparation and catalytic performance of lipases encapsulated in sol-gel materials.

Article abstract of DOI:10.1271/bbb.66.221

Three kinds of lipases (from Candida antarctica, Pseudomonas cepacia, and Pseudomonas fluorescens) were encapsulated in inorganic matrices by the sol-gel method in order to synthesize chiral compounds by kinetic resolution. Sol-gel lipases prepared with vinyltriethoxysilane had higher hydrolysis activity for 2-octyl acetate than those with other silane precursors: tetramethoxysilane, methyltrimethoxysilane, and propyltrimethoxysilane.

Full text of DOI:10.1271/bbb.66.221

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Preparation and Catalytic Performance of Lipases
Encapsulated in Sol-Gel Materials
Katsuya KATOa, Yuefa GONGa, Takao SAITOa & Yoshiyuki YOKOGAWAa
a Bio-functional Ceramics Group, Ceramics Research Institute, National Institute of
Advanced Industrial Science and Technology (AIST) 2266-98 Anagahora, Simoshidami,
Moriyama-ku, Nagoya 463-8560, Japan
Published online: 22 May 2014.
To cite this article: Katsuya KATO, Yuefa GONG, Takao SAITO & Yoshiyuki YOKOGAWA (2002) Preparation and Catalytic
Performance of Lipases Encapsulated in Sol-Gel Materials, Bioscience, Biotechnology, and Biochemistry, 66:1, 221-223,
DOI: 10.1271/bbb.66.221
To link to this article: http://dx.doi.org/10.1271/bbb.66.221
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Biosci. Biotechnol. Biochem., 66 (1), 221–223, 2002
Preliminary Communication
Preparation and Catalytic Performance of Lipases Encapsulated
in Sol-Gel Materials
Katsuya KATO,† Yuefa GONG, Takao SAITO, and Yoshiyuki YOKOGAWA
Bio-functional Ceramics Group, Ceramics Research Institute, National Institute of Advanced Industrial Science
and Technology (AIST), 2266-98 Anagahora, Simoshidami, Moriyama-ku, Nagoya 463-8560, Japan
Received August 28, 2001; Accepted October 16, 2001
prochiral compounds.5,6) Reetz et al. reported7,8) that
Three kinds of lipases (from Candida antarctica,
Pseudomonas cepacia, and Pseudomonas ‰uorescens
)
lipases entrapped in hydrophobic sol-gel materials
prepared from a mixture of tetramethoxysilane and
were encapsulated in inorganic matrices by the sol-gel
method in order to synthesize chiral compounds by
kinetic resolution. Sol-gel lipases prepared with
vinyltriethoxysilane had higher hydrolysis activity for 2-
octyl acetate than those with other silane precursors:
propyltrimethoxysilane catalyze the hydrolysis of p-
nitrophenyl propionate in aqueous solution well and
that entrapped lipases could be recycled many times
with little decrease in hydrolytic activity. However,
less attention has been paid to the eŠects of the sol-
gel encapsulation on lipase enantioselectivity. Very
tetramethoxysilane,
methyltrimethoxysilane,
and
propyltrimethoxysilane.
recently, Banjic et al. reported9) the enantioselective

Key words: lipase; enantioselectivity; sol-gel; encap-
aminolysis of a-chloroester by Candida cylindracea
sulated; vinyltriethoxysilane
lipase encapsulated in a sol-gel matrix prepared by a
modiˆcation of Reetz's procedure. There have been
few reports on the reactivity and enantioselectivity of
lipases encapsulated in sol-gel matrices. During our
study of lipase immobilization on inorganic materials
for the preparation of optically active compounds,10)
we found that lipase encapsulated in a sol-gel matrix
prepared from vinyltriethoxysilane had good reactiv-
ity and high thermal stability. Details of the reactions
of the sol-gel lipases tested follow.
The sol-gel process is a method for preparing inor-
ganic oxide matrices of metals and semimetals by
direct hydrolysis and polycondensation of active
monomeric precursors. Resulting matrices have large
surface areas; high porosity, inertness, and stability
in the presence of chemical and physical agents; and
visible and UV optical clarity. The reactions occur at
room temperature, which permits organic and bioor-
ganic molecules to be trapped within the forming sili-
ca network. Biomolecules, which are strongly encap-
sulated within the matrix and cannot diŠuse out,
generally retain their activities, gain higher stability,
and are able to react with ligands that diŠuse into the
highly porous matrix. Improved stability of the en-
trapped biomolecules and the physical and chemical
properties of the matrix are major reasons to use sol-
gels for the immobilization of biomolecules in gener-
al and for the encapsulation of enzymes in particular.
The sol-gel chemical route to various materials there-
fore has been studied very intensively in recent years,
resulting in the production of many biomolecules
that have diverse applications.1–4)
The test lipases were encapsulated in sol-gel
matrices by modiˆcation of the method described
previously.7,9) Vinyltriethoxysilane (1.14 g, 6 mmol)
was added to a mixture of 400
ml of Candida
antarctica lipase (SP 525 L, 0.8 mg protein ml,
W
15,000 units g), 100
m
l of 1 N aqueous sodium
l of aqueous poly (vinyl alcohol) (4
w w, mw 22,000), and 164 l of distilled water in a
W
‰uoride, 200
m
z
m
W
test tube. After being vigorously stirred in a vortex
mixer for 10 s, the mixture was shaken by hand for
another 5 min. The sealed tube was kept at room
temperature overnight. The material was air-dried at
room temperature for 3 days and then ground in a
mortar. After being shaken with distilled water, the
solid material was ˆltered out, and washed with cold
During the past decade, numerous articles have
been published on the use of lipases as catalysts in
organic synthesis. Reactions of commercial value
catalyzed by lipases include hydrolysis, as well as es-
teriˆcation and transesteriˆcation in nonaqueous me-
dia. Lipases also have been used to resolve kinetically
or to synthesize chiral products from racemic or
acetone and
n-hexane. The gel was air-dried over-
night and ground in a mortar, aŠording a sol-gel
lipase powder (395 mg).
In the general procedure for lipase-catalyzed
hydrolysis, the sol-gel lipase (10 mg) or free lipase
±
(10 ml) was added to a mixture of ( )-2-octyl acetate
†
To whom correspondence should be addressed. Fax: +81-52-736-7405; E-mail: katsuya-kato
＠
aist.go.jp
Abbreviations: VTES, vinyltriethoxysilane; TMS, tetramethoxysilane; MTMS, methyltrimethoxysilane; PTMS, propyltrimethoxysilane

222
K. KATO et al.
Fig. 1. Relative Hydrolytic Activity of 2-Octyl Acetate 1 with Sol-Gel Lipase SP 525.
A mixture of 1 (5.0 mg) and lipase (10 mg for sol-gel, 10 l for free) in 0.1 phosphate buŠer (pH 7.0, 2.7 ml) and acetone (0.3 ml)
was stirred at 30 for 1 h. VTES, vinyltriethoxysilane; TMS, tetramethoxysilane; MTMS, methyltrimethoxysilane; PTMS,
m
M
9
C
propyltrimethoxysilane. The highest hydrolytic activity observed with free lipase is deˆned as the 100
relative activity.
z activity for calculation of the
1 (5 mg) in 0.1
acetone (0.3 ml). After being stirred on a reciprocal
shaker at 120 rpm and at 30 C for an adequate
M
phosphate buŠer (pH 7, 2.7 ml) and
9
period, the mixture was ˆltered, and the ‰uid ˆltrate
was extracted three times with ethyl acetate. The or-
ganic layer was dried over anhydrous magnesium
sulfate, and ˆltered. The ˆltrate was concentrated in
vacuo. The residue was put through GLC in a chiral
stationary phase column (CP-cyclodex B 236M,
×
0.25 mm 30 m) under the following conditions:
detector, FID; carrier gas, He; column temperature,
130
Retention times were
11.3 min; ( )-2-octyl acetate (
)-2-octyl acetate ( )-1, 14.0 min.
Figure 1 shows relative hydrolysis of ( )-1 by
variously prepared SP 525 sol-gel lipases. The sol-gel
lipase prepared from vinyltriethoxysilane (VTES)
had the highest hydrolytic activity, approximately
9
C; injection and detector temperature, 180
9
C.
RS )-2,
S )-1, 13.3 min; and
(
RS )-2-octanol
(
Fig. 2. Comparison of the Thermal Stabilities of Sol-Gel Encap-
sulated Lipase SP 525 ( ) and Free Lipase ( ).
A mixture of sol-gel lipase (10 mg) or free lipase (10
0.1 phosphate buŠer (pH 7.0, 2.7 ml) and acetone (0.3 ml)
was heated at each temperature for 1 h. After cooling, 1
(5.0 mg) was added to the mixture and the mixture was stirred at
S


(
R
R
m
l) in
±
M
9
30 C for 1 h.
15–20
propyltrimethoxysilane
(5 1, v v) described by Reetz et al
z
higher than that from
a mixture of
and tetramethoxysilane
measured by the hydrolytic reaction of 2-octyl
acetate at 30 C after incubation with no substrate at
adequate temperatures for 1 h. This sol-gel lipase
retained approximately 70 of the initial enzymatic
activity after incubation at 90 C, but the remaining
activity of the free lipase was only 20 after incuba-
tion at 60 C. These results suggest that surrounding
7)
.
Reactivities of
9
W
W
the sol-gel lipases prepared with alkylsiloxane
(methyltrimethoxysilane, MTMS) and tetramethoxy-
silane (TMS) are very low. The sol-gel material
without lipase prepared by a similar procedure had
no hydrolytic activity. Sol-gel preparation from
VTES is very attractive because it is not an exother-
mic process during the NaF-catalyzed reaction.
Because gelation proceeds very slowly, it is not neces-
z
9
z
9
the enzyme with a sol-gel matrix markedly increases
the thermal stability of the lipase. Moreover, the sol-
gel lipase SP 525 could be recycled at least 10 times
sary to cool the reaction mixture at 4
9C to avoid a
for hydrolysis of 2-octyl acetate at 30
86 of the hydrolytic activity remained after recy-
cling 10 times (data not shown).
9C, because
decrease of lipase activity. Furthermore, alkenyl-
trialkoxysilane, which has a reactive terminal double
bond, can be transformed into unique inorganic-
organic materials by chemical methods.11)
z
Sol-gel lipases were prepared from VTES with
Pseudomonas ‰uorescens lipase (AK, Amano,
We next investigated the thermal stability of sol-gel
(VTES) lipase SP 525 (Fig. 2). Lipase activity was
18,500 units g) and Ps. cepacia lipase (PS, Amano,
W
W
30,000 units g) by a similar procedure to that

Lipases Encapsulated in Sol-Gel Materials
223
Table 1. Enantioselectivity and Reactivity for 1 with Lipases En-
In conclusion, lipase encapsulated in a sol-gel
matrix prepared from vinyltriethoxysilane had high
hydrolysis activity in aqueous solution and was ther-
mally stable. Moreover, the enantioselectivities of
lipases were increased by encapsulation in sol-gel
matrices. Further investigations of the enantioselec-
tivity and reactivity of lipases encapsulated in sol-gel
matrices are in progress.
capsulated by the Sol-Gel Methoda
b
Lipases
Time Conv (
z
)
Acetate (
z
e.e.)
E c
Sol-gel SP 525
Free SP 525
Sol-gel AK
Free AK
Sol-gel PS
Free PS
1
1
18
18
3
38
45
18
31
33
48
71
97
17
25
44
53
À
À
100
100
9
5
22
6
3
Acknowledgments
a
Sol-gel lipase (10 mg) or free lipase (10
m
l, SP 525: 0.8 mg protein ml;
W
AK: 0.7 mg protein ml; PS: 0.8 mg protein ml) was added to a solution
W
W
of 1 (10 mg) and acetone (0.3 ml) in 0.1
M
phosphate buŠer (pH 7,
We are grateful to Novo Nordisk Bioindustry
(Chiba) and Amano Pharm. Co. (Aichi) for the gifts
of the lipases. Y. G. is grateful for funding from the
STA fellowship program.
2.7 ml) at 30
9
C.
Conversion rate (
Enantiomeric ratio (
b
c
z
) was measured by chiral GC.
) was calculated as in reference 12.
E
described above. Enantioselective hydrolysis of these
lipases was examined (Table 1). Both the sol-gel SP
525 and free lipases had excellent enantioselectivity
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