Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic

media
Miguel Arroyo, Jose Mara Sanchez-Montero, and Jose Vicente Sinisterra
Department of Organic & Pharmaceutical Chemistry, Faculty of Pharmacy, Universidad Complutense, Madrid, Spain
Covalent immobilization of C. antarctica lipase B (CALB) on sepharose, alumina, and silica was undertaken. The thermal stability of these covalently immobilized catalysts were studied and compared to adsorbed derivatives from Novo Nordisk at 50C under wet conditions. Native enzyme and Novozym 435 follow a deactivation model E 3 E1 whereas covalently immobilized derivatives and SP435A follow the model E 3 E1 3 E2. This different behavior is related to the nature of the support and the immobilization methodology. Water absorption isotherms of dry solid biocatalysts in air or isooctane were used to predict the optimum preequilibrium aw value to obtain the highest rate in the esterification of (r,s)-ibuprofen. 1998 Elsevier Science Inc.
Keywords: Lipase; immobilization; stability; ibuprofen; water activity

Introduction
Lipase B from Candida antarctica (CALB) is an interesting lipase with potential application in a number of industrial processes such as the synthesis of triglycerides,2 esterification of terpenic alcohols,3 etc. Adsorbed CALB on different supports has also proven to be very regioselective in the esterification of sugars,4 nucleosides,5 and steroids,6 and very enantioselective in the resolution of secondary alcohols via hydrolysis7 or esterification in organic solvents.8 One of these derivatives, called SP435A, has been employed successfully in the preparation of pure S( )-2arylpropionic acids with antiinflammatory effect.9 In the current paper, several immobilization methods and supports have been tested for the covalent bonding of pure lipase B from C. antarctica. We have also compared the
1

thermal stability of our covalent immobilized derivatives with those obtained by Novo Nordisk by absorption of the same lipase on different polymers. All the biocatalysts were tested in the hydrolysis of triacetin due to the low activity of this lipase in the hydrolysis of triglycerides with long-chain fatty acids.10 Afterwards and at different initial water activity (a w), the best immobilized derivatives catalyzed the stereospecific esterification of racemic ibuprofen in isooctane in order to find a method to predict the best pre-equilibrium a w of the whole system to perform the reaction.

Materials and methods Materials

Native lipase B from C. antarctica (SP525) and the same lipase immobilized on Lewatit OC 1600 (SP435A) and Lewatit E (Novozym 435) were kindly supplied by Novo Nordisk Bioindustrias (Madrid, Spain). Tresylated sepharose 4B and epoxy-activated sepharose 6B were purchased from Pharmacia (Uppsala, Sweden). Silica (Kiesegel 60, size 0.015 0.040 nm, average pore

Address reprint requests to Dr. J. M. Sanchez-Montero, Universidad Complutense de Madrid, Facultad de Farmacia, Dept. Organic & Pharmaceut. Chemist., 28040 Madrid, Spain Received 22 January 1998; revised 7 April 1998; accepted 28 April 1998

Enzyme and Microbial Technology 24:312, 1999 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

0141-0229/99/$see front matter PII S0141-0229(98)00067-2

Papers
Table 1 Immobilization of lipase B from C. antarctica Immobilizatione (%) 44 18.4 42.5 27.4 90.2 48.8 9.3 g

Derivative CALB-ES-1 CALB-ES-2 CALB-TS-1 CALB-TS-2 CALB-S-1 CALB-S-2 CALB-AL-1

a

Activated support Epoxyactivated sepharosea Epoxyactivated sepharosea Tresylated speharoseb Tresylated sepharoseb Silica-TCTc Silica-TCTc Alumina-TCTd

g lipase added g 1 support 1,500 5,000 1,500 5,000 2,000 6,700 6,700

( g lipase 1 support) 660 920 640 1,370 1,810 3,250 620

C.E.f (%) 49 49 72 72 38 31 27

S.E.A.g 32 45 47 100 62 86 14

19 40 eq oxyrane groups ml 1 wet gel Grade of activation not supplied by Pharmacia LKB c 0.24 g TCT g 1 silica d 0.19 g TCT g 1 alumina e Referred to the amount of CALB added in the immobilization process f Catalytic efficiency in the hydrolysis of triacetin (see MATERIALS AND METHODS) g Specific enzymatic activity expressed as mol acetic acid released min 1 g
b

dry derivative

diameter 95 , surface area 239 m2 g 1), alumina (Aluminum 60, size 0.063 0.200 nm, average pore diameter 60 , surface area 166 m2 g 1) and isooctane (analytical grade) were obtained from Merck (Darmstadt, Germany). 2,4,6-trichloro-1,3,5triazine was supplied by Aldrich (Steinhiem, Germany). Pure triacetin was purchased from Sigma Chemical Co. (St. Louis, MO). (r,s)-2-((4-isobutyl)-phenyl)-propionic acid (ibuprofen) was donated by Boots Pharmaceuticals (Nottingham, UK).

Hydrolysis assay
As standard assay, the hydrolysis of pure triacetin was performed in 1 mm Tris-HCl buffer pH 7.0 at 37C. The acetic acid released was continuously titrated to constant pH with the help of a pHstat (Crison model microTT 2022). Several NaOH solutions (110 mm) were used as titrating agents. The catalytic efficiency of immobilized derivatives was determined as the ratio between the enzymatic activity of 3 g native lipase and the activity of the amount of immobilized derivative which contained 3 g enzyme, taking into account the percentage of immobilized enzyme (Table 1).

Protein determination
The protein content of SP525 (0.1 mg protein mg 1 derivative) was determined by the Biuret method.11 In a typical experiment, 0.1 ml of a 125 mg ml 1 SP525 solution was mixed with the Biuret reagent and the protein concentration was determined spectrophotometrically at a wavelength of 545 nm using a calibration curve of seroalbumin.

Thermal stability assays

The thermal stability assays were performed with the same amount of lipase: native or immobilized. The storage stability of native and insolubilized enzymes was performed at 50C in 0.1 m Tris-HCl buffer pH 8.0. These experimental conditions were selected as the extreme conditions. After incubation for different times, the remaining activity was measured in the hydrolysis of triacetin as described above.

Covalent immobilization on inorganic supports

The activation of silica and alumina was performed according to the 2,4,6-trichloro-1,3,5-triazine (TCT) method previously described in the immobilization of Candida rugosa lipase.12 The immobilization of lipase B from C. antarctica was performed at 4C for 6 h with low stirring. Each support (1 g) was added to different concentrations of enzyme in 10 ml of standard buffer (0.1 m Tris-HCl buffer pH 8.0). After the desired contact time, the insoluble enzyme derivative was filtered and washed with standard buffer. The percentage of immobilized enzyme was determined by the difference between the initial activity of the native enzyme solution and the activity of the filtrate after the immobilization process.

Measurement of water absorption isotherms and pre-equilibration of the reaction media

Pure lipase or immobilized derivative (100 200 mg) were previously predried with P2O5. The a w values of solid preparations were measured at 25C using a hygrometric sensor (Rotronic Hygroscopic D.T.) precalibrated with two saturated salt solutions at aw 0.11 and 0.98. The isotherms in isooctane were measured with the same amount of solid plus 1 ml of dried solvent. Enzyme preparations (native or immobilized lipases) and reaction media were equilibrated at 25C in separate containers with the help of the hygrometric sensor. Equilibration at different a w was achieved by adding the required amount of water to reach the desired a w.

Covalent immobilization on organic supports

Epoxy-activated sepharose 6B (2 g) was mixed with 10 ml of the enzymatic solution of native CALB in 0.1 m Tris-HCl pH 8.0 buffer. The mixture was stirred at 4C for 6 h and then filtered and washed with 3 10 ml of buffer solution. The immobilized biocatalyst on tresyl-activated sepharose 4B was prepared by an experimental methodology equivalent to that described above.

General procedure for esterification

The reaction media was composed of isooctane (5 ml), racemic ibuprofen (66 mm), and l-propanol (66 mm). The reaction was started by mixing different amounts of pre-equilibrated immobilized lipase to the pre-equilibrated solution to the desired a w of the system. The reactions were performed at a fixed temperature by stirring in 25 ml flasks for a specific time. The solution (100 l)

Electron microphotographs
Electron microphotographs were taken with a Zeiss DSM 940 scanning electron microscope.

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was added to 1.4 ml of isooctane and the ester conversion was analyzed by gas chromatography.9

Gas chromatographic analysis

Gas chromatography was performed in a Shimadzu GC-14A gas chromatograph equipped with a FID detector, a split injector (1:2), and a SPB-1 sulfur column (15 m 0.32 mm; Supelco, Bellafonte, PA). Injector temperature was 200C and the detector temperature 350C; the carrier gas was nitrogen. Conditions for quantitative analysis were: column temperature of 180C and N2 stream of 12 ml min 1. An external standard method was used to quantify the remnant acid and the formed ester.

Results and discussion Covalent immobilization of pure lipase B from C. antarctica

We tested different activated organic and inorganic supports for the covalent immobilization of pure lipase B from C. antarctica. As organic supports, we employed epoxy and tresylated sepharoses which differed in the length of the hydrocarbon chain attached to the matrix. As inorganic supports, we employed silica and alumina activated by the TCT methodology. The results of covalent immobilization of CALB on these activated supports are shown in Table 1. Comparing the results obtained with epoxy-activated (CALB-ES-1) and tresylated sepharose (CALB-TS-1), we may conclude that the length of the spacer arm does not affect the amount of immobilized lipase (640 660 g CALB g 1 support, 42 44% immobilization) in contrast with the results of Shaw et al.13 who observed higher immobilization values for C. rugosa lipase (CRL) with spacer arms longer than two carbons. Our results could be related to the smaller size of CALB (M w 33.3 kDa)1 14 compared to CRL (M w 60.0 kDa); however, there is a significant effect on the catalytic efficiency of the immobilized lipase. We expected higher catalytic efficiency in CALB-ES-1 (49%) where the enzyme should have larger conformational freedom due to the longer spacer arm of the epoxy-activated sepharose (12 carbons). Instead CALBTS-1, where the enzyme is closely attached to the matrix, showed higher catalytic efficiency (72%). We tried to explain this behavior taking into account the difference between the chemical bonds on different sites of the enzyme. Epoxy-activated sepharose is able to link proteins through the hydroxyl, amino, and thiol groups of their amino acids whereas tresylated sepharose only links through the amino and thiol groups, then an interaction between the oxirane group of the sepharose and the hydroxyl group of serine 105 of the catalytic triad could be involved in some extent during the immobilization. This fact could have diminished the catalytic efficiency of CALB-ES derivatives with respect to the CALB-TS. If the amount of added lipase is increased in the immobilization on both activated sepharoses, the g enzyme bonded to the support is increased as well, but the percentage of immobilization decreases (Table 1) and the catalytic efficiency remains the same (72% in CALB-TS derivatives and 49% in CALB-ES). Similar results have
Figure 1 Electron microphotograph of CALB-S-2 (original magnitude 2,000X). Enzyme aggregates of lipase B from C. antarctica can be appreciated on the surface of silica

also been observed in the immobilization of C. rugosa lipase on agarose-activated by tosylation methodology.15 The enzyme loading in the derivatives on silica is increased with the amount of lipase added in the immobilization process (CALB-S-1 and CALB-S-2); nevertheless, lipase molecules form multilayers on the support surface (Figure 1) and, as a consequence, the catalytic efficiency diminishes (3138%, Table 1). Similar results were reported in the immobilization of lipase from C. rugosa12 on TCTactivated silica. Finally, we can assume that alumina is not an adequate support for the immobilization of CALB according to the poor catalytic efficiency value of CALB-AL-1 (Table 1). The hindering effect of the pore diameter of the support (95 in silica, and 60 in alumina) can explain how the lipase loading is higher in CALB-S-2 (3,250 g g 1 support) than in CALB-AL-1 (620 g g 1 support). This indicates that the enzyme (whose size is 30 40 50 )1 is located in the external surface of alumina; therefore, a poor enzyme loading is observed. Lipase multilayers are also formed and the catalytic efficiency of both derivatives is similar (31% and 27%, respectively, Table 1).

Thermal stability
The covalently immobilized derivatives can be stored at 4C for at least two months without appreciable loss of catalytic activity. The storage stability of native and covalently immobilized lipase B from C. antarctica was studied under wet conditions at 50C. The thermal deactivation curves have been explained following the deactivation model proposed by Henley and Sadana.16 This model involves enzymatic states (E, E1, and E2) where k 1 and k 2 are first-order deactivation rate coefficients and 1 and 2 are the ratios of specific activities E1/E and E2/E, respectively [Eq. (1)]. The experimental plots of residual activity versus 5

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Table 2 Thermal deactivation of native and immobilized lipase B from C. antarctica at 50C Derivative Native CALB CALB-ES-1 CALB-ES-2 CALB-TS-1 CALB-TS-2 CALB-S-1 CALB-S-2 SP435Ae Novozym 435f
a

A1a 98.9 43.6 57.8 52.8 27.6 12.5 74.0 46.7 101

k 1 (h

1 a

A2a 0 56.0 43.3 47.2 73.3 87.8 24.6 0 0

k2 (h

1 a

b 1 2

(A3)

t1/2 (h)c 0.5 4 2.5 2.5 4.5 24 2.5 0.5 14

Fd 1 8 5 5 9 48 5 1 28

1.30 1.80 0.70 1.01 0.56 0.85 0.42 4.10 0.05

0 0.03 0.06 0.01 0.08 0.02 0.01 0 0

0 55 40 47 63 86 23 53 0

0 0 0 0 0 0 0 52.6 0

Parameters (in %) from the fitted exponential decay equation 1 and 2 are the ratio in (in %) of specific activities E1/E and E2/E, respectively c Half-life d Stabilization factor e S.E.A. 147 mol acetic acid min 1 g 1 SP435A f S.E.A. 238 mol acetic acid min 1 g 1 Novozym 435
b

storage time were adjusted to exponential decays [Eq. (2)] (single or double, with or without offset) with the help of the Simfit program developed by Dr. Bardsley.17 From the data of the adjusted equations and using Eq. (3), we could calculate all the parameters (k 1 , k 2 , 1 , 2 ), the half-life of the biocatalyst, and the stabilization factor (F, considered as the ratio between soluble and derivatives half-lives) (Table 2). o1 2 k k E3 E3 E2
1 2

2 1

1 1

k2

k1

k2

k1

k2t

(3)

(1) A 2e
1 1 k 2t

A A

A 1e

k 1t

A3
2 2

(2) k k1 e
k 1t

100

k2

k1

k2

At 50C, native CALB deactivation followed a single exponential decay which belongs to the classical first-order deactivation pattern (Table 2) in which 1 0, 2 0, and k2 0. Like native lipases A and B from C. rugosa,18 CALB loses its activity in only one step, but just a little slower. The difference between the half-life values of CALB (0.5 h) and C. rugosa pure lipases (approximately 0.25 h) may be related to the number of SOS bonds in their structure which maintains the protein integrity. CALB has three disulfide bridges in the protein structure:1 cys22-cys64, cys216-cys258, and cys293-cys311 whereas both isoforms of pure CRL have only two:14 cys60-cys97 and cys268-cys277.

Figure 2 Thermal stability of covalent immobilized derivatives of lipase B from C. antarctica at 50C

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Figure 3 Thermal stability of SP435A and Novozym 435 at 50C

As a consequence, CALB would be more thermostable than CRL isoforms with respect to the thermal deactivation by conformational change. The deactivation of covalently bonded CALB followed a double exponential decay when the derivatives were stored at 50C (Figure 2). In all cases, k 1 k 2, 1 100% and 0 (Table 2). The small values of k 2 mean a good 2 stabilization of enzymatic state E 1 . The remaining activity of the intermediate E 1 , expressed as 1, is similar in all sepharose derivatives (ES or TS series). There is not a strong relationship between this value and the enzymatic loading. The hydrophilic nature of sepharose protects the enzyme independently from the length of the spacer arm, giving similar 1, half-life values and deactivation profiles. In the derivatives prepared with silica, the activity of the intermediate state E1 may be affected by the lipase loading. The intermediate of CALB-S-1 has higher activity ( 1 86%) than the intermediate of CALB-S-2 ( 1 23%) due to the formation of lipase multilayers in the second derivative as mentioned above. As a consequence of this enzymatic aggregation, the lipase is weakly linked in the outer layers and it is quickly deactivated. We can conclude that covalent immobilization of pure lipase B from C. antarctica produces an appreciable stabilization of the biocatalyst, changing its deactivation profile. This change from a single exponential decay (native lipase) to a double one (immobilized lipase) was also observed in pure lipases from C. rugosa.18 Finally, we compared the stability of our biocatalysts with Novozym 435 and SP435A, both prepared by the adsorption methodology using different polymeric resins. These adsorbed derivatives showed a very different deactivation pattern (Figure 3) compared to our covalently im-

mobilized derivatives (Figure 2). This fact may be related to the support structure and the immobilization method. The support of SP435A derivative is a polymeric resin (Lewatit OC 1600) whereas Novozym 435 is prepared with a macroporous acrylic resin (Lewatit E). After a quick deactivation, SP435A keeps its residual activity ( 1 53%) for a long time (Figure 3). The deactivation fits model 3

Figure 4 Electron microphotograph of SP435A (original magnification 200X). Lipase B from C. antarctica is located inside the micropores of the support (Lewatit OC 1600)

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against temperature and k 1 diminishes from 1.3 h231 (native enzyme) to 0.05 h 1 (Novozym 435). From the data of Tables 1 and 2, we can conclude that SP435, Novozym 435, and CALB-S-1 are the most interesting biocatalysts due to their high activity and stability.

Water absorption isotherms and enzymatic activity

Water plays an important role in enzyme structure and function. If enzymes are used in organic media, little water is necessary in the solvent to activate the enzyme.19 When the enzyme is immobilized, the affinity of the support for water may influence its catalytic activity. The amount of water absorbed by solid carriers can be predicted from the aquaphilicity of the support.20 This property may affect the enzymatic activity in the synthesis in organic media.21 The availability of water to the biocatalyst to maintain its enzymatic activity varies depending on the water partitioning among all the components of the system: the organic solvent, the enzyme, and the solid support. The problem can be simplified using thermodynamic water activity (a w). At equilibrium, a w values will be the same for all the components in the system. In esterification reactions, lipases sometimes show an optimal initial activity at a certain content of water in the reaction medium but their behavior is different depending on the source of lipase,22 the support, and the solvent.23 Our aim was to find a general method to predict the optimum pre-equilibrium water activity of the whole system to achieve the best catalytic activity of an immobilized lipase in organic media. The adsorption isotherms of native CALB in air or isooctane are shown in Figure 6. The deviation between the water absorption isotherms of the solid in the solvent and in air produces a divergence point (D.P.). This point shows the

Figure 5 Electron microphotograph of Novozym 435 (original magnification 200 ). Lipase B from C. antarctica can be appreciated on the surface of the bead (Lewatit E)

described by Henley and Sadana, with a very high-stabilized intermediate E1 (k 2 0). This stabilization could be explained by the lipase location inside the micropores of the support where the enzyme is protected against alterations of the microenvironment (Figure 4). On the contrary, CALB is completely exposed to the medium in the surface of the small beads of Novozym 435 (Figure 5) so the deactivation model of the derivative is similar to the native lipase ( 1 0, 2 0, and k 2 0); nevertheless, CALB is stabilized

Figure 6 Adsorption water isotherms of native CALB. Conditions for measurement: 100 mg of CALB (in air); 100 mg of CALB included in 1 ml of isooctane (in solvent)

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Figure 7 Esterification of ( R , S )-ibuprofen catalyzed by native CALB in isooctane pre-equilibrated at different a w values. Conditions: 66 mM acid, 66 mM lpropanol in 5 ml of isooctane; 37C; 20 mg of SP525 ml 1; 300 rpm

lowest water activity at which we must pre-equilibrate the reaction mixture to achieve active molecules of lipase in the organic medium. The continuing similarity of the air and water isotherm from a w 0 to the divergence point (a w 0.45) suggest that this hydration range corresponds to gradual solvation of the remaining polar groups of the enzyme; however, this does not mean that the water

monolayer coverage and the hydrophobic regions of the lipase will remain exposed to the solvent.24 As a consequence, pre-equilibration of the reaction mixture at a w D.P. gave low esterification rates (Figure 7). Up to a w 0.45, the divergence between the solvent and air isotherms becomes increasingly large and a gradual increase in esterification rate was observed due to a general reorientation of

Figure 8 Adsorption isotherms of CALB-S-1. Conditions for measurement: 200 mg of CALB-S-1 (in air); 200 mg of CALB included in 1 ml of isooctane (in solvent)

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Table 3 Effect of a w on the activity of immobilized lipase B from C. antarctica Water activity ( a w) 0.10 0.40 0.60 0.75 0.85 0.90 1.00 0.25 0.45 0.80 0.90 1.00 0.65 0.85 1.00 Initial esterification rate ( mol ibuprofen propyl ester formed h 1) 9.2 14.6 13.7 16.3 151.2 181.5 99.0 3.9 9.8 11.0 89.4 17.9 1,562 2,200 3,806

Derivative CALB-S-1a

CALB-TS-1b

P435Ac

Conditions: 66 mM ibuprofen, 66 mM 1-propanol in 5 ml isooctane; 37C, 300 rpm a 200 mg of CALB-S-1 ml 1 isooctane b 30 g of CALB-TS-1 ml 1 isooctane c 50 mg of SP435A ml 1 isooctane

the polar side chains to the most favorable conformation for catalytic function. Effectively, pure lipase displayed high activity at a w values D.P. If the initial a w of the system is higher than 0.8, a monolayer of water is placed around the enzyme and the esterification rate is far higher (Figure 7). Similar results were observed for pure lipases from C. rugosa25 in the same reaction. Because water is formed in the esterification, the water activity is not controlled throughout the reaction. After 20% conversion, the progress

curve of the reaction pre-equilibrated at an initial a w 0.35 changes its shape and there is a sudden increase in the esterification rate and yield. This activation effect is explained by the water production during the reaction,26 which increases the water activity of the system to an a w value D.P. which in turn increases the reaction rate. The immobilized derivative CALB-S-1 adsorbs a lower amount of water g 1 sample than pure lipase as could be expected from the hydrophobic nature of silica (Figures 6 and 8). The continuing similarity of the CALB-S-1 absorption isotherms (in air or isooctane) up to very high values of a w (Figure 8) is related to the low affinity of silica for water whose hydrophobicity is not strongly reduced by the presence of isooctane. In fact, we could check that the behavior of silica in air or isooctane was very similar (data not shown). Evidently, the lipase immobilized on silica included in isooctane is slightly more hydrophobic than the derivative itself and, as a consequence, the adsorption isotherm with the solvent was lower than the isotherm in air. Like native lipase, after pre-equilibration of the reaction mixture at aw values higher than the divergence point (aw 0.6), we obtained active lipase molecules to start the esterification reaction. The highest esterification rate and yield were achieved with the system pre-equilibrated at aw 0.85 and 0.9 (Table 3). These values are very close to the P point which is considered as the cross point of the extension of the second slope of the absorption isotherm in isooctane and the x-axis. Native lipase also displayed its best catatalyic activity at its P point (Figures 6 and 7). Up to aw P, the esterification rate decreases which may be a result of water acting as a substrate in hydrolysis of the acylintermediate.27 The absorption water isotherms of lipase immobilized on tresylated sepharose (CALB-TS-1) are shown in Figure 9. In this case, lyophilized CALB-TS-1 was used to make the

Figure 9 Adsorption isotherms of CALB-TS-1. Conditions for measurement: 100 mg of CALB-TS-1 (in air); 100 mg of CALB-TS-1 included in 1 ml of isooctane (in solvent)

10

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Figure 10 Adsorption isotherms of SP435A. Conditions for measurement: 100 mg of SP435A (in air); 100 mg of SP435A included in 1 ml of isooctane (in solvent)

isotherm. Obviously, the affinity of sepharose for water is very high compared to silica (Figures 9 and 8, respectively). The absorption isotherm of CALB-TS-1 included in isooctane was lower than in air but no divergence point was observed; thus, the shape of the water isotherm depends on the nature of the support of the biocatalyst. The highest esterification rate was achieved at an a w 0.9 (Table 3). When higher initial system a w values were used (a w 1), the matrix was strongly hydrated and so is the microenvironment of the immobilized lipase. As a consequence, the esterification reaction was thermodynamically disfavored with respect to the hydrolysis reaction. Finally, the high hydrophibicity of the polymeric resin (Lewatit OC 1600) explains how SP435A reaches an a w 1 with very few milligrams of water (Figure 10) compared to the other supported biocatalysts. In addition, the water adsorption isotherms in air or isooctane are very close together so neither the support nor the solvent are supposed to strip water off from the enzyme. Under these conditions, lipase B from C. antarctica is always hydrated and active with a very small amount of water. Once again, we achieved the highest esterification rate pre-equilibrating the system at an a w near the P point (Table 3). The low esterification rates achieved with CALB-S-1 and CALB-TS-1 with respect to native lipase and SP435A shown in Table 3 could be expected from the low specific enzymatic activity of these biocatalysts (Tables 1 and 2).

high temperature under wet conditions (Figure 3), and a very easy methodology to be prepared. In addition, our methodology based on the P point to predict the optimum a w value to achieve the highest esterification rate in organic media is valid for different enzymes (crude and pure lipases A and B from C. rugosa25,28 and lipase B from C. antarctica) in native or immobilized form (adsorbed or covalently bonded on different supports).

Acknowledgments
Financial support from the Ministerio de Educacion y Ciencia of Spain (PB93-0469) and from the Comunidad Autonoma de Madrid (AE 00232/94) are appreciated.

List of symbols
aw 1,
2

CALB CALB-AL-1 CALB-ES-1 and 2 CALB-S-1 and 2 CALB-TS-1 and 2

Conclusions
Finally, we can conclude that SP435A is the best biocatalyst for many reasons: higher activity than the covalent immobilized lipase (in hydrolysis and synthesis), an interesting deactivation profile with a very stable intermediate state at

CRL E, E1, E2 F

Water activity Ratios of specific activities E1/E and E2/E, respectively [Eq. (1)] Lipase B from C. antarctica CALB immobilized on TCT-activated alumina CALB immobilized on epoxy-activated sepharose CALB immobilized on TCT-activated silica CALB immobilized on tresyl-activated sepharose Lipase from C. rugosa Enzymatic states during thermal deactivation [Eq. (1)] Ratio between soluble and immobilized lipase half-lives 11

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Papers
k1, k2 TCT First-order deactivation rate coefficients Trichlorotriazine
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