Novel sol-gel lipases by designed bioimprinting for continuous-flow kinetic resolutions

Article abstract of DOI:10.1002/adsc.201100329

The bioimprinting effect in sol-gel immobilization of lipases was studied to develop efficient novel immobilized biocatalysts with significantly improved properties for biotransformations in continuous-flow systems. The bioimprinting candidates were selected systematically among the substrate mimics already found in the active site of experimental lipase structures. Four lipases (Lipase AK, Lipase PS, CaLB and CrL) were immobilized by a sol-gel process with nine bioimprinting candidates using various combinations of tetraethoxysilane (TEOS), phenyltriethoxysilane (PhTEOS), octyltriethoxysilane (OcTEOS) and dimethyldiethylsilane (DMDEOS) as silica precursors. The biocatalytic properties of the immobilized lipases were characterized by enantiomer selective acylation of various racemic secondary alcohols in two different multisubstrate systems (mixture A: a series of alkan-2-ols rac-1a-e and mixture B: heptan-2-ol rac-1f and 1-phenylethanol rac-1g). Except with Lipase AK, the most significant activity enhancement was found with the imprinting molecules already found as substrate mimics in X-ray structures of various lipases. The synthetic usefulness of the best biocatalysts was demonstrated by the kinetic resolution of racemic 1-(thiophen-2-yl)ethanol (rac-1h) in batch and continuous-flow systems. Copyright

Full text of DOI:10.1002/adsc.201100329

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DOI: 10.1002/adsc.201100329
Novel Sol-Gel Lipases by Designed Bioimprinting for
Continuous-Flow Kinetic Resolutions
Gabriella Hellner,a,b Zoltꢀn Boros,c Anna Tomin,c and Lꢀszlꢁ Poppec,*
a
Department of Microbiology and Biotechnology, Corvinus University of Budapest, H-1118 Budapest, Somlꢁi fflt 14-16,
Hungary
b
Bunge Europe Innovation Centre, DÜP II. Building G. 3rd Floor, H-1097 Budapest, Illatos fflt 38, Hungary
Department of Organic Chemistry and Technology, and Research Group for Alkaloid Chemistry of the Hungarian
c
˝
Academy of Sciences; Budapest University of Technology and Economics, H-1111 Budapest, Muegyetem rkp. 3, Hungary
Fax : (+36)-1-463-3697; phone: (+36)-1-463-3699; e-mail: poppe@mail.bme.hu
Received: April 27, 2011; Revised: July 18, 2011; Published online: September 5, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100329.
Abstract: The bioimprinting effect in sol-gel immobi- mer selective acylation of various racemic secondary
lization of lipases was studied to develop efficient alcohols in two different multisubstrate systems
novel immobilized biocatalysts with significantly im- (mixture A: a series of alkan-2-ols rac-1a–e and mix-
proved properties for biotransformations in continu- ture B: heptan-2-ol rac-1f and 1-phenylethanol rac-
ous-flow systems. The bioimprinting candidates were 1g). Except with Lipase AK, the most significant ac-
selected systematically among the substrate mimics tivity enhancement was found with the imprinting
already found in the active site of experimental molecules already found as substrate mimics in X-
lipase structures. Four lipases (Lipase AK, Lipase ray structures of various lipases. The synthetic useful-
PS, CaLB and CrL) were immobilized by a sol-gel ness of the best biocatalysts was demonstrated by
process with nine bioimprinting candidates using var- the kinetic resolution of racemic 1-(thiophen-2-yl)e-
ious combinations of tetraethoxysilane (TEOS), phe- thanol (rac-1h) in batch and continuous-flow sys-
nyltriethoxysilane (PhTEOS), octyltriethoxysilane tems.
(OcTEOS) and dimethyldiethylsilane (DMDEOS)
as silica precursors. The biocatalytic properties of the Keywords: continuous flow technique; enzyme catal-
immobilized lipases were characterized by enantio- ysis; immobilization; imprinting; lipases; sol-gels
Introduction
lyze various other types of reactions such as esterifica-
tion, transesterification. Furthermore, lipases are
Synthetic application of novel biocatalytic methods is enantioselective catalysts useful in the synthesis of
a continuously growing area of chemistry, microbiolo- natural products, pharmaceutical intermediates, fine
gy and genetic engineering, due to the fact that bio- chemicals, food ingredients and bio-lubricants.[7,8,9,10]
catalysts are selective, easy-to-handle and environ-
Although the flow-through approach could provide
mentally friendly.[1–3] The broad substrate tolerance several advantages such as facile automation, repro-
and unique catalytic performance of lipases (glycerol ducibility, safety, and process reliability, it is still quite
ester
hydrolases,
triacylglycerol
hydrolases usual that chemists are working on a fixed, batch-
EC 3.1.1.3.) have attracted growing interest. Lipases based infrastructure, especially in research and devel-
are characterized by the phenomenon called interfa- opment. There are only a few examples of hydrolase-
cial activation. The active site is covered by a lid catalyzed enantioselective processes carried out in
when lipases are dissolved in water. This closed form continuous-flow systems.[11–13] Most of the continuous-
is inactive for many lipases. When lipases are in con- mode biocatalytic syntheses of chiral pharmaceutical
tact with an interface between water and an apolar intermediates are performed on a relatively large
phase, the lid opens allowing access to the active scale using immobilized lipases in a packed-bed reac-
site.[4–7]
tor.[13,14] Recently, continuous-flow packed-bed bio-
Lipases under reverse hydrolytic conditions are reactors were used to study the effects of tempera-
able to form ester bonds which enables them to cata-
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Gabriella Hellner et al.
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ture, pressure and flow rate on lipase-catalyzed kinet- structural basis with substrate-like inhibitors.[36] Since
ic resolutions.[15,16]
then 121 lipase structures have been deposited in the
The catalytic activity, selectivity, specificity and Protein Data Bank (Brookhaven PDB). Among the
enzyme stability are key factors affecting the efficien- 76 lipase structures containing ligands, those struc-
cy of biocatalysts.[17,18] Immobilization can enhance tures which included non-covalently bound ligands
the key properties of the biocatalysts such as stability mimicking
the
(Figure 1).[37–42]
The non-covalently bound ligands mimicking the
substrates
were
analyzed
and convenience of recovery and reuse.[19–24]
Entrapment of enzymes in inorganic/organic hybrid
polymer matrices (sol-gel encapsulation) has proven arrangement of substrates in the experimental struc-
to be an easy and effective way to immobilize en- tures can be divided into three categories. As expect-
zymes, whole cells, antibodies and other proteins.[25,26] ed, free fatty acids were found within lipases in the
The seminal works of Avnir,[26] Reetz[27] and their co- close proximity of the catalytically active Ser (stearic
workers led to the generalization of this technique in- acid in Figure 1 (A) and oleic acid in Figure 1 (B).
volving the acid- or base-catalyzed hydrolysis of tet- Non-ionic surfactants occupying the active site
[25]
raalkoxysilanes Si(OR)4
in the presence of an were also found in lipases [hydroxyACHTUNGTRENNUNG
enzyme. Variation of the silane precursors of different
hydrophilicity enabled further alteration of enzyme
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
performance such as activity, stability or selectivi- polyethylene glycol part of the non-ionic surfactant
ty.[27,28] Moreover, additives such as polyethylene Triton X-100 was present in a thermoalkalophilic
glycol (PEG), crown ethers or solid supports were ap- lipase [Figure 1 (E)]. Pentaethylene glycol was also
plied to modulate the pore size and increase the per- found as a substrate mimic in lipase A from Candida
meability of the substrate through the pores and the antarctica [Figure 1 (F)].
accessibility of the enzyme.[20,21,27]
Because it was expected that the best imprinting
One of the most successful strategies for enhancing molecule may vary from one lipase to another, nine
enzyme activity in organic solvents involves tuning imprinting candidates were selected for this systemat-
the enzyme active site by molecular imprinting with ic study including substrate [olive oil (OA)], products
substrates or their analogues.[29] The history of molec- [lauric acid (LA) and oleic acid (OA)], non-ionic sur-
ular imprinting is traced back to the work of Dickey factants [polyethylene glycol dodecyl ether (BRIJ-30)
in the 1940s who was inspired by Paulingꢃs theory on and polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-
the formation of antibodies.[30] Combining imprinting phenyl ether (Triton X-100)] and polyethylene glycol
with protein surface coating and salt activation was derivatives [tetraethylene glycol (TEG), polyethylene
reported as dual bioimprinting.[31] Pretreatment of a glycol 400 (PEG 400) and 1000 (PEG 1000), and the
lipase with chiral template substrates such as (R)-(À)- dilaurate of tetraethylene glycol (L-TEG-L)].
2-octanol resulted in enantioselective activation.[32]
The imprinting properties of the rationally selected
However, there are only a few publications on the additives were tested with four different lipases of
mutual effects of sol-gel entrapment and bioimprint- well documented properties such as CaLB and CrL
ing for lipases.[27,33] Imprinting effects of ad hoc select- from yeasts and Lipase AK and Lipase PS from bac-
ed molecules involving lauric acid (LA),[33] Tween terial sources. Whereas CrL is the more sensitive to
80[27] and other molecules (18-crown-6,[27] methyl-b-cy- conformation changes, Lipase AK, Lipase PS and es-
clodextrin[27]) were investigated so far.
pecially CaLB are more heat resistant and, respective-
Our goal is to investigate the imprinting effect of ly, less mobile. The most dramatic imprinting effects
rationally selected molecules (compounds which were were expected in the case of the conformationally
found as substrate mimics in experimental X-ray sensitive lipases.
structures of lipases and their structural analogues) in
Studies on subtilisin revealed that the decreased
sol-gel process resulting in robust immobilized biocat- flexibility of the enzyme relative to the aqueous situa-
alysts for continuous-flow kinetic resolutions.
tion in an apolar solvent resulted in “ligand-induced
memory”.[43] It was expected also that encapsulation
of the lipases can “freeze” their conformation and
therefore an encapsulated lipase can “remember” the
imprinting effect even after removal of the template
molecule. Thus, selection of suitable imprinting addi-
tives might be crucial to improve the activity of the
immobilized enzyme.
Results and Discussion
Rational Selection of the Imprinting Molecules for
Lipases
In 1990, the first 3D structures of lipases from Rhizo-
In a study on sol-gel immobilization of lipases, en-
mucor miehei[34] and human pancreatic lipase[35] were hancing effects of various small molecules (Tween 80,
elucidated. In 1993, the interfacial activation of the 18-crown-6 and methyl-b-cyclodextrin) and support-
lipase from Rhizomucor miehei was rationalized on a ing materials such as Celite were investigated simulta-
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Novel Sol-Gel Lipases by Designed Bioimprinting for Continuous-Flow Kinetic Resolutions
Figure 1. A–F: Crystal structures of lipases including substrate analogues within the active site. For better visibility, the cata-
lytic triad is coloured (Ser: magenta, His: blue, Asp/Glu: orange) and the amphiphile present in the active site is repeated in
the left down corner; A) Archaeoglobus fulgidus lipase with stearic acid (PDB code: 2ZYI)[37]; B) Thermomyces lanuginosa
lipase with oleic acid (PDB code: 1GT6)[38]; C) porcine pancreatic lipase-colipase with hydroxy
ACTHNUTRGNEU(GN ethyloxy)(triethyloxy)octane
(PDB code: 1ETH)[39]; D) Candida antarctica lipase B with methylpenta(oxyethyl) heptadecanoate (PDB code: 1 LBT)[40]
ACHTUNGTERNUNNG ;
E) bacterial thermoalkalophilic lipase with Triton X-100 (PDB code: 2W22)[41]; F) Candida antarctica lipase A with penta-
ethylene glycol (PDB code: 3GUU).[42]
neously.[27] However, the enhancing effects by small
molecules and solid supports in sol-gel immobilization
systems may be different. The enhancing effect in
supported sol-gel techniques is mainly due to forma-
tion of a thin enzyme layer on the large surface of the
supporting material, so lowering the diffusion limits
which are present in large enzyme aggregates.[28]
Small molecules, as additives, can induce conforma-
tional changes contributing mostly to the enhance-
ment of enzyme activity. The additives may also im-
prove the porosity of the forming sol-gel matrix thus
mitigating diffusion constraints.[24]
This study was designed in non-supported, binary
sol-gel systems (TEOS:PhTEOS 1:1)[28] solely to
detect the imprinting effect of the additives (Table 1).
Multisubstrate Kinetic Resolution as Test System for
the Preliminary Investigations
In order to distinguish effects due to induced confor-
mation changes of the enzyme and the various hydro-
phobicities of encapsulating matrices, tests of the im-
printed biocatalysts were designed with multisubstrate
mixtures consisting of a series of racemic, aliphatic al-
cohols of various chain lengths (Figure 2). Another
Figure 2. Normal and multiple kinetic resolutions with sub-
strate mixtures for testing the various biocatalysts in batch
(A) and continuous-flow (B) mode [" =pump; ~ ~ =tem-
mixture was applied to compare the behaviour of an perature control unit]
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Table 1. Screening the imprinting effect of additives in sol-gel immobilization of lipases using binary sol-gel system (TEOS:
PhTEOS=1:1). Specific activity (UB) and enantiomer selectivity (E) of the free and immobilized lipases are shown with
octan-2-ol rac-1b (Panel A) and with 1-phenylethanol rac-1g (Panel B). Reaction times were 2 h for Lipase AK, Lipase PS
and CaLB and 8 h for CrL (for further reaction details, see Experimental Section).
Form (Additive)[a]
Lipase AK
UB [UgÀ1]
Lipase PS
UB [UgÀ1]
CaLB
UB [UgÀ1]
CrL
UB [UgÀ1]
E[b]
E[b]
E[b]
E[b]
Panel A (rac-1b)
native[c]
23
23
12
20
17
19
20
11
5
7.8
6.2
4.0
5.1
4.6
5.0
5.2
4.2
2.1
4.4
4.3
3
7.1
10.7
10.4
9.2
9.0
11.3
9.2
5.7
10.2
5.7
48
9
>100
@200
>200
>200
>200
>200
>200
@200
>200
>200
>200
2.2
0.7
0.4
0.7
0.4
0.5
0.5
2.3
0.3
2.4
0.5
1.9[d]
1.8[d]
2.2[d]
2.0[d]
1.9[d]
1.8[d]
2.0[d]
1.7[d]
1.9[d]
1.6[d]
1.8[d]
SG (À)
23
25
21
20
23
19
11
24
6
SG (LA)
11
15
10
12
16
22
17
21
18
SG (OA)
SG (TEG)
SG (PEG 400)
SG (PEG 1000)
SG (BRIJ)
SG (L-TEG-L)
SG (TRX100)
SG (olive oil)
Panel B (rac-1g)
native[c]
17
15
11
6.4
31
31
30
26
26
32
29
30
29
30
14
>200
>200
>100
>200
>100
>200
>100
>100
>100
>100
>100
13
31
32
30
31
31
31
25
31
17
26
>200
@200
@200
@200
@200
@200
@200
>200
@200
>200
@200
126
16
14
19
8
20
13
25
10
17
15
@200
>200
@200
@200
>200
>200
>200
@200
>200
@200
@200
1.6
0.7
0.2
0.5
0.3
0.5
0.5
2.2
0.3
2.4
0.5
2.4
3.7
3.8
3.6
3.9
3.1
3.1
3.5
3.5
3.2
3.7
SG (À)
SG (LA)
SG (OA)
SG (TEG)
SG (PEG 400)
SG (PEG 1000)
SG (BRIJ)
SG (L-TEG-L)
SG (TRX100)
SG (olive oil)
[a]
Sol-gel immobilized enzyme with or without additive (0.5 v/v%).
If not stated otherwise, formation of the (R)-esters were preferred.
[b]
[c]
The corresponding commercially available native lipase powder for Lipase AK, Lipase PS and CrL. For CaLB, the com-
mercial liquid enzyme (Novozym CaLB L) was immobilized on Novozym carrier (1 mg carrier to 8 mL enzyme solution).
The formation of (S)-2b is preferred.
[d]
aliphatic and aromatic secondary alcohols directly. es. For this study, imprinting Lipase PS and CrL with
According to our best knowledge, no investigations PEG400 as the additive was selected (Figure 3).
on the biocatalytic properties of sol-gel entrapped en-
zymes have been performed with multisubstrate sys- strate, the Michaelis constants for various substrates
tems. for lipases are in the 0.1–40 mM range (Km =2.2 mM
Depending on the nature of the lipase and sub-
Because the lipases may be sensitive to variations and Km =5.1 mM for lipase from porcine pancreas
in the nature and area of the forming aqueous-organic with sunflower oil and palm oil, respectively;[44] Km =
interface, the sol-formation steps (mixing the silane 0.15 mM for lipase from Pseudomonas aeruginosa on
precursors with the aqueous phase, time of enzyme p-nitrophenyl palmitate;[45] Km =38 mM for lipase A
addition and NaF initiator, ultrasonic parameters) from Candida antarctica with p-nitrophenyl buty-
were varied with two enzymes (Lipase PS and CrL) rate;[8] Km =0.22 mM for pancreatic lipase on p-nitro-
in a series of preliminary experiments.
phenyl acetate[4,7]). Comparing the concentration of
In the case of CrL, intensive emulsion formation the PEG 400 with the Km values of the substrates
was crucial and thus ultrasonication had a beneficial leads to the hypothesis that the first increase in activi-
effect on the activity of the resulting biocatalyst. In ty for both sol-gel entrapped lipases is related with
contrast, variations in the sequence of sol-formation the substrate analogue-like behaviour. The second in-
resulted in no significant alterations the properties of crease in the specific activity (Km =5–20 mM interval)
the immobilized biocatalysts from Lipase PS.
can be related to the mutual effects of the substrate
Initial experiments were then performed to investi- analogue-like habitat and the impact of the additive
gate the effect of the amount of additive in the sol-gel on the quality of matrix.
formation on the properties of the encapsulated lipas-
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Novel Sol-Gel Lipases by Designed Bioimprinting for Continuous-Flow Kinetic Resolutions
to a much large activity enhancement, none of the im-
printing additives could achieve further improve-
ments.
This result can be explained in two ways. The first
being that Lipase AK is not sensitive to imprinting.
Another explanation – which we believe more plausi-
ble – is that PhTEOS or its partially hydrolyzed deriv-
atives play the same role [Figure 4 (B)] as the im-
Figure 3. Dependence of the specific activity (UB, UgÀ1) on
the concentration of PEG 400 as imprinting additive in
TEOS:PhTEOS 1:1 system (Lipase PS: 2 h; and CrL: 8 h).
Figure 4. Illustration of interfacial activation based molecu-
lar imprinting of lipase with amphiphile (A) or with silane
precursors (B)
In a work on porcine pancreatic phospholipase A2
imprinted with octyl-b-d-glucopyranoside, a similar
two-stage activation was observed.[30] The second acti- printing additives [Figure 4 (A)]. This assumption is
vation was attributed to the appearance of interfaces, supported by the fact that lipases can accept alkoxysi-
and activity increase was also saturable at higher con- lanes as substrates as indicated by the study on a
centrations.
lipid-coated lipase which was able to catalyze the oli-
Based on these considerations, the further investi- gomerization of DMDEOS.[47] Accordingly, phenyl-
gations have been performed at 10–20 mM concentra- triethoxysilane at high concentrations can occupy the
tions of the designed imprinting molecules.
active site of the lipase [Figure 4 (B)] and additives at
lower concentrations could not achieve further im-
printing effects on this enzyme.
Imprinting Effect of the Selected Additives on
Lipases Entrapped in a Sol-Gel System
The sol-gel entrapment resulted in very efficient
immobilized Lipase PS biocatalysts [SG (À)] with re-
markably higher effective specific activity than the
To evaluate the imprinting effects in this sol-gel im- free Lipase PS [UB =31 UgÀ1 vs. 13 UgÀ1, respectively,
mobilization of lipases, specific activities (UB),[28] ac- with rac-1g]. This remarkable efficiency increase is
tivity yields (YA)[28] and enantiomer selectivities (E)[46] even more pronounced when the activity yields are
from multisubstrate test reactions were compared (for taken into account [e.g., 25-fold increase for SG (À)
UB and E see Table 1; further data such as activity with rac-1b, see Supporting Information]. Similarly to
yields (YA) are given in the Supporting Information). Lipase AK, it might be hypothesized that phenyltrie-
It is worth noting that our sol-gel entrapped biocata- thoxysilane or its derivatives possess an imprinting
lysts even without imprinting additives were superior effect on Lipase PS during the sol-gel entrapment.
to the corresponding commercially available sol-gel However, a slight additional imprinting effect on
preparations (data not shown).
Lipase PS was observed with lauric acid as additive
In the case of Lipase AK, the sol-gel entrapment [UB =25 UgÀ1 for SG (LA) vs. UB =23 UgÀ1 for SG
resulted in robust immobilized biocatalysts with the (À), with rac-1b]. This is in agreement with the previ-
same effective specific activity as the free lipase ous results from the Burkholderia cepacia lipase in
powder. This represents more than 2.2-fold increase
a
sol-gel system from methyltrimethoxysilane
in the activity of Lipase AK upon immobilization (MTMOS) and tetramethoxysilane (TMOS) precur-
(YA =224%, in sol-gel matrix without additive). sors, yielding an LA-imprinted entrapped lipase with
Whereas the sol-gel immobilization of Lipase AK led 47.9- and 2.5-fold increases in specific activity over
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Gabriella Hellner et al.
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Table 2. Comparison of lipases imprinted with the best performing additives as immobilized biocatalysts in binary and terna-
ry sol-gel matrices. Specific activity (UB), activity yield (YA) and enantiomer selectivity (E) of the free and immobilized
forms of lipases in multiple kinetic resolutions are shown in Panel A (for octan-2-ol rac-1b) and Panel B (for 1-phenyletha-
nol rac-1g). The reaction times were 0.5 h for Lipase AK, Lipase PS and CaLB and 4 h for CrL in hexane:THF 2:1 solvent
(for further reaction details, see Experimental Section).
Silane matrix[a]
(enzyme
amount)
Lipase AK[b]
Lipase PS[b]
CaLB[b]
YA
[%]
CrL[b]
YA E[c]
UB
YA
E[c]
UB
YA
[%]
E[c]
UB
E[c]
UB
[UgÀ1]
[%]
[UgÀ1]
[UgÀ1]
[UgÀ1]
[%]
Panel A (rac-1b)
[d]
–
34
42
111
118
12
100
274
835
409
145
327
5.2
4.3
9.4
12.0
5.8
5.6
4
30
54
120
59
100
4.3
7.1
6.7
11.0
8.3
11.0
186
63
85
92
79
91
100
>200
@200
>200
@200
>200
>200
10.5
2.8
0.4
0.9
0.6
1.1
100 2.3[e]
Ph (1”)[f]
1854
3741
3760
6063
5222
2571
2829
1459
4611
2095
37
43
49
2.0[e]
1.5[e]
1.8[e]
OcPh (1”)[f]
OcPh (2”)[g]
PhDM (1”)[f]
PhDM (2”)[g]
Panel B (rac-1g)
481 1.3[e]
119 1.7[e]
58
101
[d]
–
109
98
127
127
74
100
198
295
136
283
217
>200
>200
>200
>200
>200
@200
23
94
118
128
125
126
100
1080
1520
737
2363
1210
>200
@200
@200
>200
@200
@200
507
86
76
125
117
113
100
1150
843
652
2263
857
@200
@200
>200
@200
@200
@200
4.4
2.3
0.1
0.8
0.8
1.2
100 2.3
Ph (1”)[f]
75
14
3.1
5.3
OcPh (1”)[f]
OcPh (2”)[g]
PhDM (1”)[f]
PhDM (2”)[g]
165 4.6
681 9.2
126
95
6.1
[a]
Ph: TEOS:PhTEOS 1:1; OcPh: TEOS:OcTEOS:PhTEOS 10:7:3; PhDM: TEOS:PhTEOS:DMDEOS 4:1:1.
Imprinting additives: no additive for Lipase AK, LA (0.5 v/v%) for Lipase PS, BRIJ (0.5 v/v%) for CrL, BRIJ for (0.5 v/
v%) for CaLB.
[b]
[c]
[d]
If not stated otherwise, formation of the (R)-esters were preferred.
The corresponding commercially available native lipase powder for Lipase AK, Lipase PS and CrL. For CaLB, the com-
mercial liquid enzyme (Novozym CaLB L) was immobilized on Novozym carrier (1 mg carrier to 8 mL enzyme solution).
The formation of (S)-2b is preferred.
Normal (single) enzyme loading (for further details, see Experimental Section).
Double enzyme loading (for further details, see Experimental Section).
[e]
[f]
[g]
the free and non-imprinted sol-gel lipases, respective- Scaling-Up the Imprinted Sol-Gel Lipases for
ly.[32]
Continuous-Flow Applications
In the sol-gel entrapment of lipases of a yeast
origin (CaLB and CrL), BRIJ and TRX100 had re- Lipases can be applied advantageously in continuous-
markable imprinting effects on the specific activity of flow systems.[11–16] Although the free native lipases
the immobilized enzymes [Table 2: UB =9 UgÀ1 vs. behave as enzyme powders in organic media and can
22 UgÀ1 for SG (À) and SG (BRIJ), respectively, for be used as fillings in packed-bed bioreactors,[15] one
CaLB with rac-1b; or UB =0.7 UgÀ1 vs. 2.4 UgÀ1 for problem to overcome is the relatively low activity and
SG (À) and SG (TRX100), respectively, for CrL with stability of the non-immobilized enzyme aggregates.
rac-1b]. The polyethylene glycol ether structures of Therefore, a packed-bed reactor filled with robust sol-
BRIJ 30 and Triton X-100 are highly reminiscent of gel immobilized enzyme is an appealing alternative.
the imprinting molecules found in the related experi-
A recent study indicated that lipases entrapped in
mental lipase structures [Figure 1 (C), (D), (F)]. In- sol-gel matrices from ternary silane precursor systems
terestingly, not only the specific activity but the enan- surpassed the catalytic properties of corresponding
tiomer selectivity of the sol-gel entrapped Lipase PS, immobilized biocatalysts from binary silane precursor
CaLB and CrL were enhanced to some extent.
systems.[28] It was also found that lipases in ternary
It is worthwhile to note that CrL preferred the acy- sol-gel systems with DMDEOS as one of the silane
lation of (S)-enantiomers of the aliphatic secondary precursors had even more beneficial properties.[48]
alcohols rac-1a–f. Earlier it was found that coating of Therefore, the potential imprinting candidates exhib-
CrL with surfactant molecules affects the recognition iting superior properties in the binary sol-gel system
of an alcohol substrate and changes the stereoprefer- were investigated further in ternary sol-gel systems.
ence.[30c]
The properties of the best imprinted ternary sol-gel li-
pases as biocatalysts were determined in batch and in
continuous-flow mode kinetic resolutions with multi-
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Novel Sol-Gel Lipases by Designed Bioimprinting for Continuous-Flow Kinetic Resolutions
ple substrates and with a single substrate on a prepa- batch mode and in a continuous-flow reactor. The
rative scale.
tests were performed with multisubstrate systems
First, scaling-up the sol-gel immobilization of the li- (mixtures A and B in Figure 2) and with the single
pases with the best performing imprinting additives racemate rac-1h (Figure 2). This series of investiga-
from sub-gram scale to multi-gram scale was per- tions was performed in hexane:methyl tert-butyl ether
formed with the earlier found best ternary composi- (MTBE):vinyl acetate 6:3:1, which is more compati-
tions
TEOS:OcTEOS:PhTEOS
10:7:3[28]
and ble with the usual pump sealings than system contain-
TEOS:OcTEOS:DMDEOS 4:1:1[48] (Table 2). Ex- ing THF.
pectedly, the lipases with the same additive which
The comparison of the four selected sol-gel lipases
proved to be the most effective in the preliminary in batch mode and continuous-flow mode with the
tests performed better in the ternary sol-gel systems multisubstrate systems revealed that irrespective of
than in the binary system (Table 2: no additive for the substrate (rac-1a–g) or enzyme (Lipase AK,
Lipase AK, LA for Lipase PS, BRIJ for CrL and Lipase PS, CaLB or CrL), the productivity (specific
CaLB). Although the two-fold amounts of the entrap- reaction rate, r) of the continuous-flow system always
ped lipases usually resulted in higher specific activity exceeded the corresponding value for the batch mode
(UB) of the forming sol-gel biocatalysts, the activity (Figure 5). This outcome was in agreement with previ-
enhancement was not proportional with the enzyme ous results in kinetic resolutions of single racemates
loading. In all cases, the higher activity yields (YA) with immobilized lipases in continuous-flow reac-
were observed for the ternary sol-gel systems utilizing tors.[15,16]
a lower lipase loading. Taking the specific activity
To demonstrate the synthetic capabilities of the im-
(UB) and enantiomer selectivity (E) together into ac- printed sol-gel lipases in a real kinetic resolution, the
count, the most efficient systems were obtained from enzymatic acylation of the less examined racemic 1-
the
TEOS:PhTEOS:DMDEOS 4:1:1 silane precursors The acylation of rac-1h was investigated with the four
with high lipase loading. imprinted lipases (Lipase AK without additive,
(thiophen-2-yl)ethanol rac-1h was chosen (Figure 2).
It is also obvious that the microenvironment of the Lipase PS with LA, CrL and CaLB with BRIJ) in two
resulting matrix has to be taken into consideration. ternary sol-gel systems at two lipase loadings. As the
Correlation was found between the order of specific CrL preparations performed with low selectivity, only
activity (UB) for the various members of the homolo- other three lipases were characterized further
gous series rac-1a–e and the hydrophobicity of the (Figure 6 and Table 3).
silane precursors (see Supporting Information).
The productivity (specific reaction rate, r) of these
After selecting the TEOS:PhTEOS:DMDEOS biocatalysts was consistently higher in the continuous-
4:1:1 silane precursor system with high lipase loading flow systems (empty bars in Figure 6) than in the cor-
and with the best imprinting molecules (no additive responding batch mode reactions (black bars in
for Lipase AK, LA for Lipase PS, BRIJ for CrL and Figure 6).
CaLB), the productivity (r) and enantiomer selectivity
The results with the lipases of sufficient enantiomer
(E) of the resulting imprinted lipases in kinetic reso- selectivity (E) in the kinetic resolution of rac-1h indi-
lutions with the sol-gel lipases were compared in cated also that a higher enzyme loading resulted in
Figure 5. Comparison of the productivity (specific reaction rate, r) for the best performing imprinted sol-gel lipases in batch
mode and in continuous-flow packed-bed reactor using multiple kinetic resolutions as test reactions. Representative data for
octan-2-ol, rac-1b and 1-phenylethanol, rac-1g with each biocatalysts are shown. [Lipase AK (TEOS:PhTEOS:DMDEOS
4:1:1, 2”, no additive); Lipase PS (TEOS:PhTEOS:DMDEOS 4:1:1, 2”, 0.5 v/v% LA); CaLB (TEOS:PhTEOS:DMDEOS
4:1:1, 2”, 0.5 v/v% BRIJ); CrL (TEOS:PhTEOS:DMDEOS 4:1:1, 2”, 0.5 v/v% BRIJ); solvent: hexane:MTBE 2:1, reac-
tion times: 1 h for Lipase AK, Lipase PS and CaLB, 4 h for CrL (batch), flow rate: 0.2 mLminÀ1 (flow); for further reaction
details, see Experimental Section and Supporting Information].
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Figure 6. Comparison of the productivity (specific reaction rate, r) for the best performing imprinted sol-gel lipases in batch
mode and in continuous-flow packed-bed reactor in kinetic resolution of 1-(thiophen-2-yl)ethanol rac-1h. [Lipase AK
(TEOS:PhTEOS:DMDEOS 4:1:1, 2”, no additive); Lipase PS (TEOS:PhTEOS:DMDEOS 4:1:1, 2”, 0.5 v/v% LA); CaLB
(TEOS:PhTEOS:DMDEOS 4:1:1, 2”, 0.5 v/v% BRIJ); reaction time: 1 h (batch), flow rate: 0.2 mLminÀ1 (flow) ; for fur-
ther reaction details, see Experimental Section].
more active immobilized biocatalysts (Table 3). Pre- using the non-selective CaLA in a continuous-flow re-
dictably, the same ternary sol-gel biocatalysts proved action. The fact that (S)-2h had only 92.0% ee after
to be the most efficient in the single racemate-based the same chromatographic purification indicated that
kinetic resolutions (rac-1h) which were the best in the the low enantiomeric excesses of the acetates (R)-
multiple substrate tests (TEOS:PhTEOS:DMDEOS and (S)-2h are due to the sensitivity of the product to
4:1:1, high lipase loading; Table 2).
racemization rather than the insufficient selectivity of
Finally, the two most selective sol-gel entrapped li- the lipases in the continuous-flow system.
pases (Lipase AK, 2”loading in TEOS:Ph-
TEOS:DMDEOS 4:1:1 system without additive and
CaLB, 2”loading in TEOS:PhTEOS:DMDEOS 4:1:1 Conclusions
system with BRIJ) were applied to perform the kinet-
ic resolutions of 1-(thiophen-2-yl)ethanol rac-1h in The study on bioimprinting effects of substrate-mim-
continuous-flow reactors on preparative scale icking molecules in sol-gel immobilization of lipases
[Figure 2 (B); Table 4]. Analysis of the products at with binary and ternary silane precursor compositions
various times in the 24-h-long runs indicated the op- indicated that independently from the nature of sol-
erational stability of both sol-gel biocatalysts (see gel matrix, the most pronounced imprinting effects
Supporting Information).
were found with such additives which were found
Although the enantiomer selectivity for both bio- mimicking the substrates in the experimental struc-
catalysts was reasonably high in the batch mode tests tures of the lipases.
(E>200, see Table 3), the enantiomeric excess of the
This case study revealed lauric acid as being the
isolated acetate (R)-2h from the preparative-scale most effective imprinting additive for lipase PS (the
continuous-flow experiment was not as high as in the crystal structure of lipase from Burkholderia cepacia
test reactions. In a next experiment, however, the (S)- included stearic acid) while in the cases of Candida
1h of 99.2% ee was acetylated quantitatively to (S)-2h species (CaLB, CrL) tetraethylene glycol dodecyl
Table 3. Comparison of lipases imprinted with the best performing additives as immobilized biocatalysts in ternary sol-gel
matrices in batch mode kinetic resolutions of 1-(thiophen-2-yl)ethanol rac-1h.
Silane precursors (enzyme amount)
Lipase AK[a]
UB [U8 gÀ1]
Lipase PS[a]
UB [UgÀ1]
CaLB[a]
UB [UgÀ1]
E
E
E
[b]
–
70
23
71
31
70
>100
90
89
81
@200
28
44
73
33
70
>100
>100
73
>100
>100
70
25
66
23
54
@200
>100
>200
>100
>200
TEOS:OcTEOS:PhTEOS 10:7:3 (1”)[c]
TEOS:OcTEOS:PhTEOS 10:7:3 (2”)[d]
TEOS:PhTEOS:DMDEOS 4:1:1 (1”)[c]
TEOS:PhTEOS:DMDEOS 4:1:1 (2”)[d]
[a]
Imprinting additives: no additive for Lipase AK, LA (0.5 v/v%) for Lipase PS, BRIJ for (0.5 v/v%) for CaLB; solvent:
hexane:MTBE 2:1, reaction time 1 h; for further reaction details, see Experimental Section.
The corresponding commercially available native lipase powder for Lipase AK and Lipase PS. For CaLB, the commercial
liquid enzyme (Novozym CaLB L) was immobilized on Novozym carrier (1 mg carrier to 8 mL enzyme solution).
Normal (single) enzyme loading (for further details, see Experimental Section).[d] Double enzyme loading (for further de-
tails, see Experimental Section).
[b]
[c]
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Novel Sol-Gel Lipases by Designed Bioimprinting for Continuous-Flow Kinetic Resolutions
Table 4. (S)-1-(Thiophen-2-yl)ethanol (S)-1h and (R)-1-(thiophen-2-yl)ethyl acetate (R)-2h prepared by kinetic resolutions
with sol-gel immobilized imprinted lipases in continuous-flow reactors. [rac-1h, 5 mgmLÀ1 in hexane:MTBE:vinyl acetate
6:3:1, 0.2 mLminÀ1, 308C, 24 h flow time].
Compound
Lipase (precursors, additive, enzyme amount)
Y[a] [%]
ee[b] [%]
(S)-1h
(R)-2h
Lipase AK (TEOS:PhTEOS:DMDEOS 4:1:1, -, 2”)
CaLB (TEOS:PhTEOS:DMDEOS 4:1:1, BRIJ, 2”)
35.0
20.8
99.2
88.8
[a]
Yields refer to isolated products after chromatographic separation.
The ee values were determined by enantioselective GC.
[b]
were added to the vial while continuously shaking. To com-
plete the polymerization of the product in a sol suspension,
the mixture was shaken for 12 h at room temperature. The
resulting solid was washed with IPA (7 mL), distilled water
(5 mL), IPA (5 mL) and n-hexane (5 mL). The residual
white powder was dried in a vacuum desiccator for 3 h
(until 0.4 mmHg final level of vacuum). Experiments with
Lipase PS and CrL were carried out in triplicates. The sol-
gel lipase preparations were stored at room temperature.
ether (BRIJ 30) exhibited the most significant im-
printing effect [the porcine pancreatic lipase resem-
bling similarity to CrL included tetraethylene glycol
octyl ether and the crystal structure of CaLB included
methylpentaACHTUNGTRENNUNG(oxyethyl)-heptadecanoate].
The high efficiency of sol-gel systems containing tri-
alkoxysilanes OcTEOS or PhTEOS may be explained
by assuming that these silanes or their partially hydro-
lyzed forms induce an imprinting effect as well. This
assumption can explain why Lipase AK (lipase from
Pseudomonas fluorescens) entrapped in OcTEOS- or
PhTEOS-based sol-gel matrices required no imprint-
ing additives for maximal performance.
The rational selection of imprinting molecules can
be combined with proper compositions of silane pre-
cursors in large-scale production of sol-gel immobi-
lized lipases for various applications. The robust sol-
gel entrapped forms of the selected four lipases (from
Lipase AK, Lipase PS, lipase B from Candida antarc-
tica and lipase from Candida rugosa) proved to be
ideal biocatalysts in kinetic resolution of a racemic al-
cohols in batch and continuous-flow systems.
Scaling up the Sol-Gel Immobilization of Lipases
using Ternary Silane Precursor Systems
Scale-up with the general enzyme loading (1ꢀ): The general
sol-gel production procedure with 20-fold enzyme amounts
(1 g of Lipase AK, Lipase PS and CrL; and 1 mL of CaLB
L) using ternary silane precursors [TEOS:OcTEOS:Ph-
TEOS (10:7:3) or TEOS:PhTEOS:DMDEOS (4:1:1);
60 mmol (in the given molar ratio)] was performed with the
selected imprinting additives [no additive for Lipase AK,
LA (0.5 v/v%) for Lipase PS, BRIJ (0.5 v/v%) for CrL,
BRIJ for (0.5 v/v%) for CaLB]. All the other components
of the general procedure were added in 20-fold amounts.
Scale-up with doubled enzyme loading (2ꢀ): The 20-fold
amount of enzyme (1 g of Lipase AK, Lipase PS and CrL;
and 1 mL of CaLB L) was added to a system containing 10-
fold amounts of the components of the general procedure,
the ternary silane precursors [TEOS:OcTEOS:PhTEOS
(10:7:3) or TEOS:PhTEOS:DMDEOS (4:1:1); 30 mmol (in
the given molar ratio)] and the selected imprinting additives
[no additive for Lipase AK, LA (0.5 v/v%) for Lipase PS,
BRIJ (0.5 v/v%) for CrL, BRIJ for (0.5 v/v%) for CaLB].
Experimental Section
Materials
Details on enzymes, chemicals and solvents are given in
Supporting Information.
Characterization of the Sol-Gel Lipase Preparations
Using Multisubstrate Test Systems
General Procedure for Sol-Gel Immobilization of
Lipases (using Binary and Ternary Silane Precursor
Systems)
The biocatalyst (native lipase or sol-gel immobilized lipase;
50 mg in each case) was added to the solution of multisub-
strate mixture A (50 mL; equimolar mixture of hexan-2-ol
rac-1a, octan-2-ol rac-1b, nonan-2-ol rac-1c, decan-2-ol rac-
1d and dodecan-2-ol rac-1e) or multisubstrate mixture B
(50 mL; equimolar mixture of heptan-2-ol rac-1f and 1-phe-
nylethanol rac-1g) in hexane:THF 2:1 (1 mL) or in hexa-
ne:MTBE 2:1 (1 mL) containing vinyl acetate (100 mL). The
resulting mixture was shaken at 308C in a sealed glass vial
at 1200 rpm. Samples were taken directly from the reaction
mixture (sample size: 50 mL, diluted with hexane to 500 mL)
at 0.5, 1, 2, 4, 8, 24 h, and analyzed by GC (for details see
Supporting Information).
IPA (200 mL), TRIS-HCl buffer (0.1M, pH 7.5, 390 mL), the
imprinting additive [0.5% v/v, 5 mL: lauric acid (LA), oleic
acid (OA), polyethylene glycol 400 (PEG 400), polyethylene
glycol 1000 (PEG 1000), Triton X-100 (polyethylene glycol
tert-octylphenyl ether, TRX100), BRIJ 30 (polyethylene
glycol dodecyl ether, BRIJ), tetraethylene glycol (TEG), tet-
raethylene glycol dilauryl ester (l-TEG-L) or olive oil] and
NaF solution (1M, 100 mL) were mixed in a 5-mL glass vial
and the resulting solution was shaken at 1000 rpm at room
temperature for 10 min. The corresponding silane precursors
[TEOS:PhTEOS (1:1), TEOS:OcTEOS:PhTEOS (10:7:3)
or TEOS:PhTEOS:DMDEOS (4:1:1); 3 mmol (in the given
molar ratio)] and enzyme (50 mg or 50 mL for liquid CaLB)
The specific activity of the biocatalyst [UB (mmol”
minÀ1 gÀ1)] could be determined in the test reaction from the
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Gabriella Hellner et al.
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amount of the racemic alcohol [nrac (mmol)], the conversion
(c), the reaction time [t (min)] and the total mass of the
native free enzyme or the sol-gel immobilized biocatalyst
gary. Thanks to Thales Nanotechnology Inc. for the fillable
CatCartꢁ columns.
[mB (g)] using the UB =(nrac ”c)/ACTHNUTRGNEUNG
(t”mB) equation.[28] Because
the activities in the present work were calculated from
single time-point data, the term “activity” must be consid-
ered only for comparative evaluation of the catalytic effi-
ciencies, and not for the real kinetic behaviour of the biocat-
alyst.
The activity yield [YA (%)] can be calculated from the ef-
fective specific activity of the immobilized biocatalyst
(UE,imm) compared to the specific activity of the native en-
zymes (UB,native).[28]
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