Cross-linked enzyme crystals as highly active catalysts in organic solvents

Full text of DOI:10.1021/ja960081s

5
494
J. Am. Chem. Soc. 1996, 118, 5494-5495
Cross-Linked Enzyme Crystals as Highly Active
Catalysts in Organic Solvents
form of CLEC catalysts which combines high activity and
productivity in neat organic solvents.
Enzyme activity in organic solvents is intimately related to
11
water content, the size and morphology of the catalyst particles,
and the enzyme microenvironment. These parameters can be
adjusted by preparing lyophilized complexes of enzymes with
Nazer Khalaf, Chandrika P. Govardhan, Jim J. Lalonde,
Rose A. Persichetti, Yi-Fong Wang, and
Alexey L. Margolin*
12
13
14
carbohydrates, organic buffers, and salts. However, despite
the wide use of lyophilization for preparation of enzymes for
catalysis in organic solvents, the process is not fully understood
and may cause significant reversible denaturation of enzymes.12
Since CLECs are solid crystalline particles insoluble in both
water and organic solvents, their drying can be achieved by
washing them with solutions of different surfactants in organic
solvents, thus completely avoiding lyophilization. A number
Altus Biologics Inc., 40 Allston Street
Cambridge, Massachusetts 02139-4211
ReceiVed January 10, 1996
The low activity of enzymes in organic solvents limits their
1
impressive synthetic potential. It is not unusual to see processes
employing more enzyme than substrate by weight.2 In the case
of lipases, this low efficiency can be explained, at least in part,
by the low purity of commercial preparations. For example,
the commercial preparations of two of the most synthetically
useful lipasessfrom Candida rugosa (CRL) and Pseudomonas
cepacia (LPS)scontain less than 5-6% and 1% of the lipase,
respectively. The large amount of impurities makes the
downstream processing difficult and expensive, and complicates
our understanding of the mechanistic aspects of enzymatic
catalysis in organic solvents.
1
5
16
17
of anionic, cationic, and nonionic surfactants were used
in the initial screening. The activity was measured after drying
and then repeated after 12 days storage at room temperature.18
On the basis of this screening, the best surfactants were chosen
and dry CLECs were prepared on a large scale. In a typical
1
9
experiment CLECs of LPS (30 g protein) suspended in 10
mM Tris, 10 mM CaCl2, pH 7.0, were transferred to a sintered
glass funnel (porosity ∼5 µm). The buffer above the CLECs
was decanted or removed by suction. The equal volume of
2
-butanone containing 30 g of the detergent N,N′,N′-poly-
One would think that by using lipases in purified form much
higher activity could be achieved. In addition, recent pulbica-
tions have indicated that purified lipases exhibit higher enan-
tioselectivity in hydrolytic resolutions, simply by eliminating
(oxyethylene(10))-N-tallow-1,3-diaminopropane was added to
the CLEC cake. The solvent and surfactant were removed by
gentle suction. The mixture was transferred to a fritted pressure
filter funnel after breaking up any lumps and dried in a stream
of nitrogen to a water content of about 2-3 % as determined
by Karl Fisher titration.
The activities of the resulting CLECs21 in the resolution of
alcohols, acids, and amines are presented in Table 1. Not only
are the CLECs of three different enzymes (two lipases and
subtilisin) much more active than their crude counterparts on a
weight basis (columns 4 and 5), but in all of the reactions shown
in the table their specific activity per milligram of protein is
higher as well (column 6). Thus, in order to achieve the same
activity in the resolution of menthol or sulcatol, for example,
3
,4
contaminating enzymes with opposite enantioselectivity.
Surprisingly, the potential synthetic benefits of purified lipases
in organic solvents have not been realized to date.5 Not only
is the cost of purified lipases higher, but their stability and
,6
activity in organic solvents are lower than those of their crude
counterparts.7,8
9
Recent work on cross-linked enzyme crystals (CLECs) has
demonstrated that enzymes in this form acquire high stability
1
0
while preserving their activity in high-water mixtures. By
definition, CLECs are highly purified since their preparation
includes crystallization of the enzymes. Subsequent chemical
cross-linking locks the enzymes in the crystalline form, thus
affording insolubility and stability. Here we disclose a novel
(11) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141-144. It is
accepted that effect of water on enzyme activity in organic solvents is better
described in terms of thermodynamic water activity. See: Bell, G.; Halling,
P. J.; Moore, B. D.; Partridge, J.; Rees, D. G. Trends Biotechnol. 1995, 13,
468-473. Halling, P. J. Enzyme Microb. Technol. 1994, 16, 178-206.
(12) Dabulis, K.; Klibanov, A. M. Biotechnol. Bioeng. 1993, 41, 566.
(13) Blackwood, A. D.; Curran, L. J.; Moore, B. D.; Halling, P. J.
Biochim. Biophys. Acta 1994, 1206, 161-165.
(
1) Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114-120. Poppe, L.;
Nov a´ k, L. SelectiVe Biocatalysis; VCH PUblishers: New York, 1992. Wong,
C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry;
Pergamon: New York, 1994.
(
2) For some representative examples, see: Bovara, R.; Carrea, G.;
Ferrara, L.; Riva, S. Tetrahedron: Asymmetry 1991, 2, 931-938. Wang,
Y.-F.; Lalonde, J. J.; Momongan, M.; Bergbreiter, D. E.; Wong, C.-H. J.
Am. Chem. Soc. 1988, 110, 7200-7205. Palomaer, A.; Cabre, M.; Ginesta,
J.; Mauleon, D.; Carganico, G. Chirality 1993, 5, 320-328. Gotor, V.;
Brieva, R.; Gonzales, C.; Rebolledo, F. Tetrahedron 1991, 47, 9207-9214.
Therisod, M.; Klibanov, A. J. Am. Chem. Soc. 1987, 109, 3977-3981. Njar,
V. C. O.; Caspi, E. Tetrahedron Lett. 1987, 28, 6549-6552. Burgess, K.;
Henderson, I. Tetrahedron Lett. 1991, 32, 5701-5704. Bevinakatti, H. S.;
Banerji, A. A.; Newdakar, R. V. J. Org. Chem. 1989, 54, 2453-2455.
Gutman, A. L.; Shapira, M.; Boltanski, A. J. Org. Chem. 1992, 57, 1063-
(14) Khmelnitsky, Y. L.; Welch, S. H.; Clark, D. S.; Dordick, J. S. J.
Am. Chem. Soc. 1994, 116, 2647-2648.
(15) Aerosol 22, dioctyl sulfosuccinate (AOT), Niaproof Types 8 and 4,
TEEPOL HB7.
(16) Methyltrioctylammonium chloride (Aliquat 336), N,N′,N′-poly-
(oxyethylene(10))-N-tallow-1,3-diaminopropane.
(17) Brij 30, Brij 35, Tritons X-15, X-100, and X-405, sorbitan
sesquiolerate (Arlacel 83), Span 85, Tergitols NP 4 and NP 35.
(18) The activity of the dry samples was measured in transesterification
of n-amyl alcohol with ethyl acetate in toluene for CRL and LPS and
transesterification of N-Ac-PheOEt with n-propanol in isooctane for ABL.
While several surfactants exhibited high activity right after drying, only a
few maintained this high level after storage.
1
065. Parida, S.; Dordick, J. J. Am. Chem. Soc. 1991, 113, 2253-2259.
3) Wu, S.-H.; Guo, Z.-W.; Sih, C. J. J. Am. Chem. Soc. 1990, 112,
(
1
990-1995. Hernaiz, M. J.; Sanchez-Montero, J. M.; Sinisterra, J. V.
Tetrahedron 1994, 50, 10749-10760. Ng-Youn-Chen, M. C.; Serreqi, A.
N.; Huang, Q.; Kazlauskas, R. J. J. Org. Chem. 1994, 59, 2075-2081.
(19) The preparation of CRL-CLEC was described in ref 4. LPS was
crystallized according to: Bornscheuer, U.; Reif, O-W.; Laush, R.; Fretag,
R.; Scheper, T.; Kolisis, F. N.; Menge, U. Biochim. Biophys. Acta 1994,
1201, 55-60. Alcalase (Novo) from Bacillus licheniformis (ABL) was
crystallized according to the procedure reported for subtilisin Carlsberg
(Tuchsen, E.; Ottesen, M. Carlsberg Res. Commun. 1977, 42, 407-420)
with slight modifications. CLECs were obtained by cross-linking with
(
4) Lalonde, J. J.; Govardhan, C. P.; Khalaf, N. K.; Martinez, O. G.;
Visuri, K. J.; Margolin, A. M. J. Am. Chem. Soc. 1995, 117, 6845-6852.
(
(
5) Nishio, T.; Kamimura, M. Agric. Biol. Chem. 1988, 52, 2631.
6) Yamane, T.; Ichiryu, T.; Nagata, M.; Ueno, A.; Schimozu, S.
Biotechnol. Bioeng. 1990, 36, 1063-1069. Tsai, S.-W.; Dordick, J.
9
Biotechnol. Bioeng., in press.
glutaraldehyde according to the published procedure. Cross-linked enzyme
(
7) Bovara, R.; Carrea, G.; Ottolina, G.; Riva, S. Biotechnol Lett. 1993,
crystals of CRL, LPS, and ABL are sold under the trade name Chiro-
TM
1
5, 169-174. Wehtje, E.; Aldercreutz, P.; Mattiasson, B. Biotechnol.
Bioeng. 1993, 41, 171-178.
8) Ottolina, G.; Carrea, G.; Riva, S.; Sartore, L.; Veronese, F. Biotechnol.
Lett. 1992, 14, 947-952.
9) St. Clair, N. L.; Navia, M. A. J. Am. Chem. Soc. 1992, 114, 7314-
316.
10) Persichetti, R. A.; St. Clair, N. L.; Griffith, J. P.; Navia, M. A.;
Margolin, A. L. J. Am. Chem. Soc. 1995, 117, 2732-2737.
CLEC -CR, ChiroCLEC-PC, and ChiroCLEC-BL, respectively, and are
commercial products of Altus Biologics, Inc. (Cambridge, MA).
(20) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1982, 104, 7294-7299.
(
(
(21) A similar procedure was applied to the preparation of CRL-CLECs
and ABL-CLECs. The optimal surfactants for CRL- and ABL-CLECs
were Tergitol Type TMN-6 (poly(oxyethylene glycol ether)) and Tergitol
Type 15-S-3 (poly(oxyethylene glycol ether)), respectively.
7
(
S0002-7863(96)00081-9 CCC: $12.00 © 1996 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 23, 1996 5495
Table 1. Enzyme-Catalyzed Resolutions in Organic Solvents (Rates (V) are in µmol/min‚mg at 25 °C)
V
CLEC/V crude;
a
c
3
d
3
equal proteine Et CLEC E crude
compound
menthol (449 mM)
Ibuprofen (97 mM)
actylating agent and solvent enzymeb
V
CLEC × 10
V
crude × 10
VA (449 mM); toluene
n-amyl alcohol (460 mM)
isooctane
CRL
CRL
1220
57.5
4
0.13
62
90
.100
16
7.2g
.100g
hydroxyhexanoic acid (532 mM) BuOH (1060 mM) toluene
CRL
LPS
LPS
LPS
ABL
85
15000
6700
11700
12.5
2.2
90
20
44.5
2.9
7.9
55g
.100
28
2g
.100
35
sec-phenethyl alcohol (200 mM)
sulcatol (80 mM)
VA (200 mM) toluene
VA (120 mM) toluene
VA (120 mM) toluene
TFB (400 mM) t-BuOH
1.7
3.4
2.6
3.6
2
-octanol (80 mM)
9.8
41
9.9
46
methyltryptamine (200 mM)
a
b
VA, vinyl acetate; TFB, trifluoroethyl butyrate. The water contents of crude enzyme preparations were 9.3%, 2.5%, and 5% for CRL, LPS,
c
and ABL, respectively. The water contents for CLEC were 13.3%, 2.3%, and 2.5% for CRL, LPS, and ABL, respectively. CLEC concentrations
were 4, 1, 10, 1.2, 0.4, 0.4, and 16 mg/mL. The amounts of the surfactants in the final preparations of CLECs were 16%, 40%, and 50% (w/w)
d
e
for CRL, LPS, and ABL, respectively. Crude enzyme concentrations were 20, 15, 50, 8.3, 40, 40, and 28 mg/mL, respectively. The total protein
contents in crude commercial preparations of CRL, LPS and ABL were 10%, 0.7%, and 7%, respectively. The protein content of crude lyophilized
f
ABL was 50% (w/w). Reactions rates and enantioselectivities were assayed by capillary gas chromatography on Cyclodex B, (alcohols) (R,R)Whelk-
OH (Ibuprofen) GC, and Chiracel OJ HPLC (methyltryptamine) columns. E was calculated according to ref 20. For discussion on applicability
of E for reactions affected by inhibition or catalyzed by crude enzymes see ref 4. g
Eapp was calculated as in an irreversible case on the basis of the
ee of the product at low conversion.
one will need to use about 300-fold less catalyst. The striking
difference in activities between crude enzyme preparations and
CLECs cannot be explained by differences in water content in
the case of CRL and ABL (Table 1, footnotes b and c). When
crude CRL and ABL preparations were dried to the same water
contents as those of CLECs (13.3% for CRL and 2.5% for
ABL), the activities changed by less than 12%.
lipids, celite, and other proteins. In addition, surfactants may
simply facilitate the transfer of hydrophobic substrate molecules
through the layer of tightly bound water to the binding site of
an enzyme. While much more work is necessary to elucidate
the exact mechanism of the effects of surfactants on CLEC
activity, the practical consequences of this phenomenon are
immediately clear: the high specific activity, purity, and stability
of CLECs result in the high catalyst productivity in neat organic
solvents.
2
5
To the best of our knowledge, this is the first demonstration
2
2
that pure lipases can be extremely active in organic solvents.
In addition, CRL-CLECs exhibit much higher enantioselectivity
than crude CRL preparation (Table 1, entries 1-3). In this case
the increased enantioselectivity of CRL-CLECs is clearly due
to the removal of competing hydrolases.4
In order to demonstrate the productivity of CLECs in organic
solvents on a preparative scale, we chose the resolution of sec-
phenethyl alcohol (50 mmol; 6.1 g) with vinyl acetate (50 mmol;
4
.3 g) in toluene (100 mL) catalyzed by LPS-CLECs (1.3 mg
In addition to the surfactants chosen for the preparative
procedures, several others (Tritons and AOT for CRL and AOT
and Brij 30 for ALB) exhibited high activity after storage. The
combination of several effects may account for this dramatic
increase in activity of pure lipases in CLEC form. All of our
data indicate that surfactants play a crucial role in the activity
enhancement. Indeed, when CLECs were dried to a similar
water content without surfactants, their activity was 19 (ABL),
solid; 1 mg protein). The reaction mixture was allowed to stir
at room temperature for 16 h at which time the conversion
reached 50%. The isolated yield of phenethyl acetate was 4.5
g (98.5% ee) giving the volumetric productivity of 30 g/L and
26
a substrate to catalyst ratio of 4700. The high productivity
of low molecular weight synthetic catalysts is thought to be
27
their key advantage over high molecular weight enzymes. This
example clearly demonstrates that despite the high molecular
weight and nonaqueous reaction medium, CLECs can be highly
productive and compare favorably even with the best of
synthetic asymmetric catalysts.
7
9 (LPS), and more than 100 (CRL)-fold lower than that
2
3
reported in Table 1. The presence of amphiphilic surfactants
may help to maintain a better water balance or the native
24
conformation of amphiphilic lipases, which in crude prepara-
tions can in part be achieved by different contaminants such as
JA960081S
(
22) High activity was also demonstrated for purified subtilisin soluble
(24) Broos, J.; Visser, J. W. G.; Engbersen, J. F. J.; Verboom, W.; van
Hoek, A.; Reinhoudt, D. N. J. Am. Chem. Soc. 1996, 117, 12657-12663.
(25) Rather than accelerating the resolution reactions in water, surfactants
slowed them down: Bornemann, S.; Crout, D. H. G.; Dalton, H.;
Hutchinson, D. W. Biocatalysis 1994, 11, 191-221.
in organics (Paradkar, V. M.; Dordick, J. S. J. Am. Chem. Soc. 1994, 116,
5
009-5010). High activity of lipase complexes soluble in neat organic
solvents was achieved only with crude lipases: Kamiya, N.; Goto, M.;
Nakashio, F. Biotechnol. Prog. 1995, 11, 270-275. Okahata, Y.; Fujimoto,
Y.; Ijiro, K. J. Org. Chem. 1995, 60, 2240-2250.
(26) The productivity reached 8000 after 72 h reaction when 100 mmol
sec-phenethyl alcohol was used.
(
23) The water content in these experiments was 13.3% for CRL-
CLECs, 1.7-2% for LPS-CLECs, and 2.2-2.4% for ABL-CLECs.
(27) Jacobsen, E. N.; Finney, N. S. Chem. Biol. 1994, 1, 85-90.