Catalytic properties of lipases immobilized on various mesoporous silicates

Article abstract of DOI:10.1271/bbb.67.203

Lipases SP525, AK, LIP, and PS were immobilized on three kinds of mesoporous silicates (FMS, PESO, and SBA) with diameters of 27 to 92 A. The amount of lipase activity adsorbed on these supports was related to the pore size of the silicate. Enantioselectivities of immobilized lipases were similar to those of free lipases, and recycling could be done in both aqueous and organic solvents.

Full text of DOI:10.1271/bbb.67.203

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Catalytic Properties of Lipases Immobilized on
Various Mesoporous Silicates
Katsuya KATOa, Roxana IRIMESCUa, Takao SAITOa, Yoshiyuki YOKOGAWAa & Haruo
TAKAHASHIb
a Bio-functional Ceramics Group, Ceramics Research Institute, National Institute of
Advanced Industrial Science and Technology (AIST) 2266-98 Anagahora, Shimoshidami,
Moriyama-ku, Nagoya 463-8560, Japan
b Toyota Central R & D Lab. Inc.
Published online: 22 May 2014.
To cite this article: Katsuya KATO, Roxana IRIMESCU, Takao SAITO, Yoshiyuki YOKOGAWA & Haruo TAKAHASHI (2003)
Catalytic Properties of Lipases Immobilized on Various Mesoporous Silicates, Bioscience, Biotechnology, and Biochemistry,
67:1, 203-206, DOI: 10.1271/bbb.67.203
To link to this article: http://dx.doi.org/10.1271/bbb.67.203
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Biosci. Biotechnol. Biochem., 67 (1), 203–206, 2003
Note
Catalytic Properties of Lipases Immobilized on Various Mesoporous Silicates
, ,
† Roxana IRIMESCU,1 Takao SAITO,1 Yoshiyuki YOKOGAWA
1,
1
Katsuya KATO
and Haruo TAKAHASHI
2
1Bio-functional Ceramics Group, Ceramics Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan
2Toyota Central R & D Lab. Inc., Nagakute, Aichi 480-1192, Japan
Received July 30, 2002; Accepted September 18, 2002
Lipases SP525, AK, LIP, and PS were immobilized
on three kinds of mesoporous silicates (FMS, PESO,
and SBA) with diameters of 27 to 92 Å. The amount of
lipase activity adsorbed on these supports was related to
the pore size of the silicate. Enantioselectivities of im-
mobilized lipases were similar to those of free lipases,
and recycling could be done in both aqueous and organ-
ic solvents.
racemates of various secondary alcohols.11,12)
The mesoporous silicates FMS (pore diameters:
27 Å, 62 Å, and 77 Å), PESO (65 Å), and SBA (92 Å)
were prepared as reported elsewhere.7,13) Lipase AK
(
(
(
Pseudomonas ‰uorescens, Amano, Nagoya), PS
Pseudomonas cepacia Amano, Nagoya), and LIP
Pseudomonas aeruginosa, Toyobo, Osaka) were
dissolved individually in 20 m phosphate buŠer (pH
7.0). The lipase solution was separated from the
undissolved materials by centrifugation and used
for the immobilization on mesoporous silica (AK;
1.1 mg of protein ml; PS, 0.8 mg of protein ml;
LIP, 1.2 mg of protein ml). The protein concentra-
M
Key words: mesoporous silica; immobilization; lipase;
enantioselective acetylation; secondary al-
cohol
W
W
W
Immobilization and encapsulation of enzymes on
solid inorganic materials have been intensively stud-
ied because of the materials' potential use with
biocatalysts and biosensors.1) Inorganic supports
with surfaces suitable for the immobilization of en-
zymes with high activity have been sought. Periodic
mesoporous materials with uniform pore diameters
of 15–300 Å have been studied;2,3) diameters in this
range are close to the diameters of enzyme molecules.
These materials might be eŠective as supports.
Uniformity of the pores of mesoporous materials
allows control of molecular adsorption based on size.
Enclosure of the protein in a well-deˆned space may
help to prevent denaturation. Moreover, the pore
structure can be changed systematically, providing
insight into changes in the tertiary structure and
tion of the solution was measured by Bradford's
method with a Bio-Rad protein assay kit (Hercules,
CA). Lipase SP525 (Candida antarctica, 0.8 mg of
protein ml, Novo, Chiba) in solution was used
W
without further puriˆcation or dilution. The lipase
immobilization method has been reported else-
where.7) Mesoporous silica powder (30 mg) suspend-
ed in 3 ml of lipase solution was kept overnight at
4
9
C with gentle stirring. The supernatant was sepa-
rated by centrifugation at 1000 for 10 min at 4 C,
g
9
and the resulting pellet was washed with cold water,
acetone, and hexane. It then was dried under reduced
pressure for 3 h and stored at 4
9
C until use in the
catalytic experiments. The hydrolysis of
p
-nitro-
phenyl caprate was used to measure the activities of
the free and immobilized lipases.
active site during catalysis. Small biomolecules,
Results of lipase adsorption on the ˆve meso-
porous silicates (FMS-27 Å, FMS-62 Å, and FMS-77
Å, PESO, and SBA) are shown in Fig. 1. Lipase
activities were adsorbed on all of them, the highest
ratio being on FMS-62 Å. However, lipase AK was
adsorbed by FMS-62 Å, FMS-77 Å, PESO, and SBA
in similar ratios. Of the lipases immobilized on the
ˆve silicates, the FMS-62 Å lipases had the highest
4,5)
cytochrome
c,
trypsin,6) horseradish peroxidase,7)
and chloroperoxidase,8) in hexagonal mesoporous
silicates have been immobilized, but to our
knowledge, no report has been made of lipase im-
mobilization in mesoporous silicates. Lipases, the
most widely used enzymes in synthetic organic che-
mistry, catalyze chemo-, regio-, and stereoselective
hydrolysis of carboxylic acid esters or the reverse
reactions in organic solvents.9,10) We compared the
use of three diŠerent mesoporous silicates for lipase
immobilization. The enantioselectivity and reactivity
of the immobilized lipases was tested by the use of
hydrolysis activities with
p-nitrophenyl caprate.
These lipases therefore were further examined for
their kinetic resolution of racemates and recycling
use.
Lipase-catalyzed acetylation of secondary alcohols
†
To whom correspondence should be addressed. Fax: +81-52-736-7405; E-mail: katsuya-kato
＠
aist.go.jp

204
K. KATO et al.
Fig. 3. Recycling of Immobilized Lipases in (a) Organic Solvent
and (b) Aqueous Solution.
After the reactions were ended, pellets were collected by cen-
trifugation, and the reactions were repeated. (
immobilized on FMS-62 Å; ( ) lipase PS immobilized on
FMS-62 Å. See the reaction conditions in the legend of Fig. 2.
Fig. 1. Adsorption Ratios of Lipases on Mesoporous Silicates.
The adsorption ratio was calculated as [1-(lipase activity in the

), Lipase SP525
supernatant solution after adsorption) (lipase activity before
W

×
adsorption)] 100 (
z). The immobilization method is described
in the text. FMS27: Mesoporous silicate FMS with a pore size of
27 Å.
stopped at about 30
free lipase SP525 had high enantioselectivities (E 14)
＝＞
z conversion. Immobilized and
100) with (R)-selectivity for all of the racemates,
1a–1e, in both acetylation and hydrolysis. The same
enantioselectivity was found for 1a–1e with immobi-
＝
＝
＝
E
lized and free PS ( 5 for 1b,
E
8 for 1a,
E
Fig. 2. Lipase-catalyzed Enantioselective Acetylation of Sec-
＞
100 for 1c, E 25 for 1e).
E
＝＞100 for 1d, and ＝
ondary Alcohols in Organic Solvent.
Enantioselectivities of AK and LIP did not change
with immobilization, evidence that the immobiliza-
tion of lipases on mesoporous silicate does not aŠect
lipase enantioselectivity.
Acetylation:
(2.0 mg), vinyl acetate (25
in diisopropyl ether (3.0 ml) was stirred at 30
mixture of racemic acetate; ( )-2a–2e (2.0 mg) and immobi-
lized lipase (2.0 mg) in 0.1 phosphate buŠer (pH 7.0, 2.7 ml)
and methanol (0.3 ml) was stirred at 30 C.
a
mixture of racemic alcohol;
l), and molecular sieves 4A (30 mg)
C. Hydrolysis: a
(R,S)-1a–1e
m
9
R,S
M
Results of the recycling of FMS-62 Å-immobilized
lipases SP525 and PS in hydrolysis and acetylation
are shown in Fig. 3. Reactions were done under the
conditions described above. Eighty-ˆve percent or
more of the original activity of lipases immobilized
on FMS remained after eight recyclings by acetyla-
tion in the organic solvent. Remaining activity was
9
was done as reported previously (Fig. 2).12) In brief,
FMS-62 Å-immobilized lipase (2.0 mg) was added to
a mixture of racemic alcohol (2.0 mg), vinyl acetate
(25
m
l), and molecular sieves 4A (30 mg) in di-
C. After being stirred
70z or more after the same number of recyclings by
isopropyl ether (3.0 ml) at 30
9
hydrolysis in aqueous solution, evidence that lipase
immobilized in mesoporous silicate is tightly ad-
sorbed in the mesopore space and is not present on
the outer surface of the particle.
for an adequate period, the reaction mixture was
ˆltered, and the ˆltrate put through GC or HPLC to
measure the conversion and enantiomeric purity of
the reacted acetate and remaining alcohol. The GC
and HPLC conditions were GC; column, CP-cyclo-
Thermal stabilities of the lipases were examined.
Free SP525 and FMS-62 Å-immobilized lipase SP525
were incubated in phosphate buŠer (pH 7.0) at vari-
ous temperatures for 1 h, and the activity remaining
×
dex B 236M (0.25 mm 30 m); carrier gas, He; detec-
tion; ‰ame ionization detector. HPLC was done with
UV detection at 254 nm in a Chiralcel OJ (Daicel,
Tokyo) column with a mobile phase of hexane-
at 30
had about 15
1 h of incubation, immobilized lipase SP525 retained
90 of its activity at the same temperature, evidence
9
C was measured. Although free lipase SP525
z
of its original activity at 60 C after
9
isopropyl alcohol (8.5 1.5; v v) at the ‰ow rate of 1
W
W
ml min. Enantioselectivities were measured by GC
z
W
for 2a–2d and by HPLC for 2e. The immobilized li-
pases were used for hydrolysis under the following
conditions: the immobilized lipase (2 mg) was com-
bined with a solution of racemic acetate (2 mg)
that the thermal stability of immobilized lipase is
greater than that of free lipase.
Nitrogen adsorption isotherms and the pore-size
distribution curve for the FMS material before and
after lipase SP525 adsorption are shown in Fig. 4.
Pore volumes of the FMS-62 Å material decreased to
about half of the original volume after lipase im-
mobilization. The pore volume for the FMS of small
pore size, 27 Å, was the same before and after lipase
in 0.1
M
phosphate buŠer (2.7 ml, pH 7.0) and
C. After being stirred for an
methanol (0.3 ml) at 30
9
adequate period, the reaction mixture was worked up
in the usual manner, and the resulting solid material
put through GC and HPLC. All lipase reactions were

Immobilization of Lipases on Various Mesoporous Silicates
205


Fig. 4. Changes in Nitrogen Adsorption Isotherms and the Pore Diameter Curve ( ) with ( ) Immobilization of Lipase SP525 on
Mesoporous Silica FMS-62 Å.
was immobilized (not shown). This shows that lipase
molecules were adsorbed in the pore spaces of FMS
material with a pore diameter of 62 Å.
tivities, and recycling could be done at least eight
times with enantioselective reactions in both aqueous
and organic solvents. Furthermore, thermal stability
of the immobilized lipases was greater than that of
the free ones. Immobilized lipases have important
practical applications, such as biocatalysts for chiral
resolution.
Preparative-scale enzymatic resolution of 1a and
1d was done by acetylation of the alcohols with vinyl
acetate in diisopropyl ether in the presence of lipases
immobilized on mesoporous silica FMS-62 Å. Im-
mobilized lipase SP (30 mg) and (R,S)-1a (200 mg)
were added to a suspension of vinyl acetate (0.3 ml),
diisopropyl ether (3 ml), and molecular sieves 4A
(30 mg), and the suspension was stirred for 15 h at
room temperature on a reciprocal shaker at 120 rpm,
and then ˆltered. The ˆltrate was concentrated by
evaporation under reduced pressure. The residue was
separated on a silica gel column, and its constituents
was eluted with ethyl acetate hexane (1 4–1 2, v v).
Acknowledgments
We thank the Amano Enzyme. Co. (Aichi) and
Novo Nordisk (Chiba) for providing the Amano AK,
PS, and Novo SP525. R. I. gratefully acknowledges
receipt of a JSPS fellowship.
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W
W
W
W
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