The Journal of
physical Chemistry ~
0 Copyright 1995 by the American Chemical Society
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VOLUME 99, NUMBER 22, JUNE 1,1995
LETTERS Activation of a Lipase Triggered by Interactions with Supercritical Carbon Dioxide in the Near-Critical Region Yutaka Ikushima,**?j* Norio SaitoJ Masahiko Arai,o and Harvy W. BlanchL National Industrial Research Institute of Tohoku, Nigatake, Miyagino-ku, Sendai 983, Japan: PRESTO, Research Development Corporation of Japan, Nagata-cho, Chiyoda-ku, Tokyo 100, Japan: Institute for Chemical Reaction Science, Tohoku University, Aobaku, Sendai 980, Japan: and Department of Chemical Engineering, University of Califomia, Berkeley, Califomia 94720 Received: October IO, 1994; In Final Form: February 22, 1995@
Secondary structures of an enzyme (Candida cylindracea) in supercritical carbon dioxide and interactions operating between carbon dioxide and enzyme molecules were studied by Fourier transform infrared spectroscopy combined with microgravimetry at pressures of 6.86-19.12 MPa and at 304.1 K. It was found that, in very limited pressure range near the critical point of carbon dioxide, the interactions are greatly increased and allowed to provoke drastic conformational changes of the enzyme, causing active sites to emerge and catalyze the stereoselective synthesis of (s>-( -)-oleic acid 3,7-dimethyl-6-octenyl ester from oleic acid, and (f)-citronellol. Supercritical carbon dioxide medium in the near-critical region should be a trigger to the activation of the enzyme by causing the movement of surface groups and creating an active site, producing a stereoselective machinery.
Introduction The interactionsbetween enzyme and solvent in supercritical fluids are of fundamental importance because small changes in pressure or temperature near the critical point may bring about great changes in enzyme-catalyzed activity.'-5 Strong effects of pressure on the rate of enzyme-catalyzed reactions in supercritical carbon dioxide (SC-CO2) have been p r e ~ e n t e d ; ~ , ~ for example, the reaction rates are significantly enhanced near the critical point. However, the influence of the conformation of active enzymes on the reactivities has not been elucidated. It has been noted that, in the near-critical regions of supercritical fluids, local d e n ~ i t i e s and ~ , ~ local compositions*-" of the
* To whom
correspondence should be addressed.
' National Industrial Research of Tohoku. t PRESTO. 5 @
Institute for Chemical Reaction Science. University of California. Abstract published in Advance ACS Abstracts, May 15, 1995.
solutions are dramatically changed, and occasionally solute/ solvent clustering formation influences reactivities.'* A study of an enzyme-catalyzed oxidation in SC-CO2 using highpressure electron paramagnetic resonance (EPR) spectroscopy revealed that aggregation of substrate molecules in SC-CO2 is greatly increased in the near-critical region.' However, the participation of the aggregation in the reaction has not been understood at the microscopic level. The industrial importance of organic syntheses catalyzed by lipases emphasizes a comprehensive understanding of their conformation and propertie~.'~ However, no studies have been carried out to elucidate the effects of nonaqueous solvents on both enzymatic specificity and stereoselectivity of lipases. It was reported that 1-terpene esters were stereoselectively synthesized by Candida cylindracea (CCL) lipase from acyl donors and secondary terpene alcohols in nonaqueous solutions, but not from primary terpene alcohol^.^^-^^ Recently, we have first
0022-3654/95/2099-8941$09.00/0 0 1995 American Chemical Society
8942 J. Phys. .Chem., Vol. 99, No. 22, 1995
Letters
Figure 1. Schematic diagram of high-pressure apparatus for FT-IR and microgravimetry measurements: ( I ) tip, (2) SUS 304 hook, (3) CCL film cast on a zinc sulfide plate, (4) zinc sulfide window, (5) feedback loop, (6) heating block, (7) C02 cylinder, (8) molecular sieve, (9) pump, (IO) back-pressure regulator.
obtained a CCL-catalyzed ester synthesis fiom a primary alcohol of (&)-citronelloland oleic acid in SC-C02 at 304.1 K.I6 The optical purity of the product was found to be very sensitive to reaction pressure; it was at least 98% enantiomeric excess (ee) at 8.41 MPa, while it was 26% ee at 7.58 MPa and 4.1% ee at 10.15 MPa. What is responsible for this sensitive stereoselectivity appearing only in the near-critical region? It is probable that some particular interactions act among the enzyme, the reactants, and the solvent in the near-critical region. The unliganded structure of a lipase from CCL was very recently determined" and indicates that the activation of the CCL requires the movement of surface groups which are likely to interact with an activated complex. In exploiting and controlling the catalytic properties of the lipase, the structure-function relation and factors that trigger the activation should be elucidated. Of our particular interest is the relationship between the conformation of active CCL in SC-C02 and its enantioselectivity in the above-mentioned reaction. What are pressure dependence of solvent clustering near the CCL molecules, influence of the cluster molecules on the conformation of the CCL molecules, participation of the conformational change in the reactivities, etc? To answer these questions, we have conducted an experimental work using Fourier transform infrared spectroscopy combined with microgravimetry. In the present work, interactions operating between SC-C02 and CCL enzyme molecules have been studied with particular attention to those in the near-critical region, where significant aggregation would occur and the stereoselectivity is endowed. Furthermore, information on the secondary structure of CCL at different pressures in SC-CO2 has been obtained by FT-IR,and then the enzyme-substrate complex has been inferred.
Experimental Section In Figure 1 is shown a schematic diagram of the experimental apparatus used for FT-IR and microgravimetry measurements. A specimen of CCL film (ca. 2 x mm thick) on an ZnS plate (1 0 mm long x 5 mm wide x 2 mm thick) was prepared by casting certain volumes of its aqueous solution of 5.0 x
M on the plate and drying in air. The specimen obtained was hung from a hook in a high-pressure cell. It was dried by flushing dry N2 for more than 24 h and then deuterated by flushing D20 vapor for 7-8 days in order to remove the HOH bending band at 1640 cm-', which would overlap amide I. After drying and deuteration at atmospheric pressure, SC-C02 was passed through the cell at about 1 MPa to remove the residual D20 vapor for 12 h. Then, while SC-C02 was passed, the pressure was increased to 19.12 MPa at the rate of 0.15 MPd min for 8.3-8.5 MPa and 0.75 MPdmin for 10-19.12 and decreased at the rate of 2 MPdmin. The measurements of FTIR and microgravimetry were simultaneously performed at increasing and decreasing pressures at a constant temperature of 304.1 K maintained within f O . l K. The pressure reading was done at a precision within fO.O1 MPa. In the microgravimetry measurement, the change in weight of the specimen was able to be measured from a vertical displacement of the hook. The displacement data were obtained at a rate of 1 time per 8.0 s from the change in the voltage applied to the cylindrical piezoelectric actuator in the constant tunneling current mode. The data were converted into the weight changes with a precision of 10 pg. In the FT-IR measurement, the interferrogram was accumulated at a rate of 1 scan per 1.2 s at 2 cm-l nominal resolution. For the spectrum collected, the band analysis was performed by Fourier selfdeconvolution and curve-fitting methods; the former method was used to determine the band peak positions and then the latter to determine the peak strengths with Lorentzian or Gaussian band profiles. The method'* used in this study to enhance the resolution of the spectrum band analysis is based on the modified Fourier deconvolution algorithm developed by Kauppinen et aI.l9 and the finite impulse response operator method formulated by Jones and Shimokoshi.20 In both FT-IR and microgravimetry, the results were referenced to those for the ZnS plate free from the CCL film at the same increasing and decreasing pressures, and the following results are after this reference.
J. Phys. Chem., Vol. 99, No. 22, 1995 8943
Letters Time I sec
a
f 30 25
-
20 15 10 -
st IH
211
b
0
100
PressureIMPa
h
80
Time I sec
$ 0 v)
73
60
2
V .* L.
W
5
7
9
11
13
15
17
19
’
40
.-5 c
E:
20
L5
0
Pressure (MPa)
Pressure I MPa Figure 2. (a) Pressure dependence of change of weight of CCL film at pressures of 6.86- 19.12 MPa at 304.1 K. a WIW represents changes in weight of CCL film relative to the initial value. (b) Changes in a W I W between 8.0 and 9.2 MPa. The rate of pressure increase was about 0.19 MPdmin.
Results and Discussion Figure 2 gives the change in weight of the CCL film with increasing pressure of SC-CO2, showing large increases at a limited range of 7.7-8.7 MPa, similarly to the above-mentioned stereoselective esterification. This indicates that the CCL takes up a large number of C02 molecules at those pressures; in other words, C02 molecules considerably aggregate into the enzyme, the mole ratio of C02 to CCL (MW = 56 000) molecules, NCOJ N E ,reaching a maximum of ca. 3000. Figure 2b further shows a fluctuation in the change of weight. It was found that, when the pressure was decreased below 7.5 MPa, the CCL film went back to the initial weight. It can be assumed, from those observations, that strong interactions operate between C02 and CCL at the particular pressures, and the interactions are not so stable and reversibly changeable with respect to pressure. Some relevant phenomena were reported in the literature. Ascarelli and Nakanishi” observed excess surface adsorption of supercritical argon on a metal in the critical region. Randolph et al.1.22reported that such surface aggregation phenomena in the near-critical region caused great increase in the reactivities of enzymatic and spin exchange reactions. The amide I bands are intimately related to the molecular geometry and ultimately determine the protein secondary structure.23Figure 3 presents FT-IR results, giving information on the secondary structure of the CCL at different pressures. The assignment of the component bands is based on FT-IR data of proteins published Four interesting facts are
Figure 3. (a) Pressure dependence of the relative intensities of a-helix at 1657 cm-l (0 with plus), irregular at 1646 cm-I (O), turn at 1666 cm-’ (0),and P-sheet-rich at 1635 and 1622 cm-I (0 with slant). X-ray: 31% a-helix, 17% P-sheet.I3 (b) Changes of intensities of absorption bands at 1722 ( a , A), 1760 (0,4). 2963 (0.H).and 2860 cm-I (V,V) during upward (open symbols) and downward (filled symbols) pressure cycles (6.86- 19.12-6.86 MPa) and of percent ee ( x ) of (S)-( -)-oleic acid 3.7-dimethyl-6-octenyl ester during the esterification from oleic acid, and (&)-citronellol.
implicit in Figure 3. ( 1 ) In the very limited pressure range between 7.7 and 8.5 MPa, the intensity of the band at 1646 cm-l due to an increase of irregular structures significantly increased, being accompanied by remarkable decrease in that at 1657 cm-’ correspondingto a decrease in a-helical structures. (2) Only in the same near-critical region, the intensities of the bands at 1722, 1760,2963, and 2860 cm-l suddenly increased, indicating the new formation of ester (R-COO-R’) or diester (R-OCO-COO-R’) species. The C02 molecules and alkyl chains of amino acids in a-helix are combined directly to produce ester species linking different chains of amino acids. The IR spectra are sharp and very active, and the species must stick out spatially toward outside of the protein surface (unfolding process). This is further supported by the increase of turn (1666 cm-’) and irregular structures and the decrease of a-helical ones (Figure 3a). (3) The spectra recorded during increasing and decreasing the pressure agreed with each other very well. This leads to more favorable formation of diester species rather than ester ones because the strength of the 0-R (or 0-R’) bonds of the former is very weak. Irreversible changes in the secondary structures of enzymes induced by increasing hydrostatic pressure have been however, the reversible pressure-induced unfolding refolding process of a protein in this study has been scarcely found out.3o (4) The pressure range where the optical purities are developed in the CCL-catalyzed esterification strikingly overlaps that for the sudden conformational changes of the CCL. The characteristic structure3’ of CCL possesses the hydrophobic tunnel which is occluded by a “lid” and present in the
8944 J. Phys. Chem., Vol. 99, No. 22, 1995
R” I
Letters
(d)
I R
I 0 G1y124NH
‘,
,
Alazlo NH ’
I
,o-- c
along the tunnel, followed by accommodation of longer acyl chain of the intermediate. Thus, it can be said that the solvent is a trigger to the activation of CCL by causing the lid movement and creating a hydrophobic active-site groove, making CCL to be stereoselective machinery. In addition, the unusual reactivity should be associated with the substrate-solvent clustering that occurs in the near-critical region as pointed out by Randolph*’ and Debenedetk3’
References and Notes -07-
I
\
Serzos
R (fatty acid) Figure 4. Schematic representation of an inferred enzyme-substrate complex. The covalently modified O;,atom of Ser 209 and oxyanion hole is shown schematically. The R”(C-C-C-C=C
