Bioconversion of phospholipids by immobilized phospholipase A2

,O”IINAL

OF

Biotechnolo ELSEVIER

Journal of Biotechnology 40 (1995) 145-153

Bioconversion of phospholipids by immobilized phospholipase

A,

Ricardo Madoery a, Clelia G. Gattone a, Gerard0 Fidelio b,* a Facultad de Ciencias Agropecuarias, Universidad National de Cbrdoba, Chrdoba, Argentina b Departamento de Q&mica Bioldgica, Facultad de Ciencias Q&micas, Universidad National de Cdrdoba, 5016~Chdoba, Argentina Received 6 January 1995; accepted 2 March 1995

Abstract Phospholipase lysocompounds.

A, selectively This bioconversion

hydrolyses the ester linkage at the sn-2 position of phospholipids forming has importance in biotechnology since lysophospholipids are strong bioemulsi-

fiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A, from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex@ support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A, was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A, was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V,,, 883.4 Fmol mg-’ min-’ and a K, 12.9 mM for soluble form and V,,, = 306 pmol mg-’ min-’ and a K, = 3.9 for immobilized phospholipase A*) were in agreement with the immobilization effect. The results obtained with CM-Sephadex@phospholipase A, system give a good framework for the development of a continuous phospholipid bioconversion process. Keywords: A. mellifera; Phospholipase

A 2; Immobilized

phospholipase;

CM-Sephadex;

Ionic binding; Phospholipid

bioconversion

1. Introduction

Abbreviations: PLA, = Phospholipase A,; CM = carboxy methyl; K, = Michaelis constant; V_ = maximum velocity; v = activity (initial rate); DEAE = diethylaminoethyl; Tris = tris(hydroxymethyl)aminomethane; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PA = phosphatidic acid; HPTLC = high performance thin layer chromatography; IE = immobilized enzyme; SL soybean lecithin; S = substrate concentration * Corresponding author.

Phospholipase A, (PLA,, EC 3.1.1.4) hydrolyses the fatty acid ester bond at the sn-2 position of 1,2-diacyl-sn-phosphoglycerides. This enzyme plays an important role in many biochemical phenomena (TronchBre et al., 1994). Frequently, PLA,s and other lipolytic enzymes act in association to membranes in biological medium (Errasfa, 1991) and immobilized phospholipase could serve as selective

probe

vivo action

(Ferreira

0168-1656/95/$09.50 8 1995 Elsevier Science B.V. All rights resewed SSDI 0168-1656(95)00040-2

for

a model

et al., 1993).

system

of the

in

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The hydrolytic reaction promoted by PLA, has importance in biotechnology since the lysophospholipid derivative products have strong bioemulsifying properties. The use of lysolipids as food additives is well known (Van Nieuwenhuyzen, 1976), and recently new applications of these compounds were reported in the pharmaceutical industry and in biomedicine (Mukherjee, 1990; Tai Lee and Cooper, 1991). Moreover, biomedical reactor with immobilized PLA, was also reported to be of potential application for hypercholesterolemia treatment (Labeque et al., 1993; Shefer et al., 1993). Immobilized enzymes (IE) catalyze specific biochemical reactions, with the advantage that they are usually more stable than soluble forms and can be reused. Immobilized phospholipase systems could also be applied in continuous phospholipid bioconversions. The most important sources of PLA, are snake and insect venoms, and pancreas and exudates of mammals. The PLA, enzyme from Apis melliferu venom is commercially available and very active. Its purification and characterization, the complete amino acid sequence and the number of disulfide bridges has been previously determined (Shipolini et al., 1971,1974a,1974b). The three-dimensional structure of this PLA, was also determined to be similar to that of bovine pancreas (Kuchler et al., 1989) and others secretory PLA,s (Scott et al., 1990a). Sephadex ion exchangers have several advantages over other solid supports: (a) a high protein adsorption capacity; (b) hydrophilicity of the supports as important factor for enzyme preservation; (c) the immobilization procedure by adsorption is simple; and (d) the starting gel matrix is easily regenerated (Porath and Axen, 1976; Birnbaum, 1993). Phospholipids are amphiphilic molecules and can exist in many different aggregated forms in water depending on the hydrophobic chain length and polar head group (Lichtenberg et al., 1983; Reynolds et al., 1991). It is expected that the bioconversion with immobilized phospholipases will depend on the substrate aggregation state, but information about IE action upon aggregated phospholipid is limited at present compared to

the knowledge about the action of soluble PLA,s (Jain and Gelb, 1991). For non immobilized forms of PLA,, several authors have discussed the heterogeneous catalytic mechanism (Tinker and Wei, 1979) and the interfacial activation effect (Scott et al., 1990b). Also, soluble PLA, activity toward mixed micelles has been studied (Dennis, 1973). The micelle aggregate provides a well-defined matrix for kinetic studies (Roberts et al., 1978). Upreti and Jain (1978) have studied the effect of phosphatidylcholine state on the hydrolysis rate by bee venom PLA,. With respect to immobilized PLA,, antecedents in the literature are scarce and some of them are related to covalently attached snake venom enzymes (Lombard0 and Dennis, 1985; Ferreira et al., 1993). Also, several PLA,s were reversibly fixed in different matrixes, but only with the objective of a further purification of the PLA, enzyme by chromatographic process (Reynolds and Dennis, 1991). In a previous work, we have immobilized pancreatic PLA, upon DEAE-Sephadex with a high fixation level but the immobilized system had low activity (Madoery and Camusso, 1991). The objective of this work was to immobilize bee venom PLA, into an ion exchanger support, and to study the kinetic behaviour of the IE on natural phospholipids as substrate.

2. Materials and methods 2.1. Reagents and chemicals

PLA, from bee venom was obtained from Sigma Chemical Co. (St. Louis, MO), and was used without further purification. The enzyme has an apparent MW of 19000 and an isoelectric point of 10.5 (Shipolini et al., 1971). A stock solution of 1.25 mg ml-’ of enzyme was prepared in 50 mM Tris-HCl pH 8.2. Carboxymethyl Sephadex C-25 (CM-Sephadex), a weakly acid cation exchanger and diethylaminoethyl Sephadex A-25 (DEAE-Sephadex) a weakly basic anion exchanger were obtained from Sigma. Both are based on a crosslinked dextran matrix with a dry bead size of 40-125 micras and a capacity of

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of Biotechnology 40 (1995) 145-153

4.0-5.0 meq g-i. Purified soybean lecithin (SL) composed of 33.4% PC, 28.6% PE, 29.6% PI and 8.3% PA (Madoery and Camusso, 1991), was used as substrate. Soybean PC was isolated from SL and purified by column chromatography using a method adapted from several techniques. Briefly, SL (5 g) was extracted with ethanol and adsorbed on Kieselgel40 Fluka (10 g) and succeseluted sively with ethanol and chloroform/methanol/water 60:40:3 (v/v). PC elutes in the last fraction. Purity was tested by HPTLC using a horizontal separating chamber (DESAGA) and Silica gel 60 chromatoplates u sin g Argentina) (M erck, chloroform/methanol/water 69:24:4 (v/v> as developing system. Product gave a single spot when iodine vapors were employed for detection. Triton X-100 was from Roehm and Haas Co. Tris and all other reagents were of analytical grade. 2.2. Enzyme immobilization CM-Sephadex C-25 (75 mg) in 1.5 ml of distilled water was treated with 0.2 N HCl (7.5 ml> for conversion to proton form and washed exhaustively with water. The support was decanted and the excess water was removed. Then, 4 ml of a diluted enzymatic solution is added (0.3 mg ml-’ in 50 mM Tris-HCl pH 8.0). The medium was allowed to mix during 30 min in a rotary system (Btichi RE-111 at 60 rpm) at room temperature. IE system was obtained by filtration and vacuum dried at 50°C. Dried IE was stored at 5°C. A similar procedure was used for the immobilization onto DEAE-Sephadex A-25 but in this case the resin was previously treated with 0.1 N NaOH (hydroxide form). The fixation level was estimated substracting the protein remaining in the supematant after binding compared to the initial protein concentration. Protein was spectrophotometrically determined at 280 nm using a Ea.‘“/” = 13--2 Ian 2.3. Enzymatic assay The enzymatic activity of either unbound enzyme or immobilized form was estimated using

147

SL/Triton X-100 ratio 1:4 mixed micelles. Assays were carried out in 50 mM Tris pH 8.4 at 40” C in presence of 5 mM CaCl,. Reaction medium (2 ml) was rotated in a rotatory system (Biichi RE1111 during 5 min at 60 rpm. Control assay was performed without enzyme. PLA, activity was obtained by microdetermination of released fatty acids according to Dole and Meinertz (1960) using ethanolic KOH and connected to a digital titrator (Digital Titrator HACH Model 16900). The lower limit of detection was 0.1 pmol of fatty acids. The values given are averages of at least duplicate assays. In some of the assays we tested the enzymatic products by HPTLC. 2.4. Reuse assay Reaction medium (2 ml with 2 mg of IE) as described in Enzymatic assay (see above) was submitted to successive cycles. After the activity was measured in the first cycle the supematant was discarded. Then the EI was washed once with distilled water. The water was discarded and the PLA, activity was measured again adding a new batch of substrate. 2.5. Apparent kinetic parameters determination Unbound enzyme (15 pg) or the equivalent amount of the immobilized form were used in the same conditions of Enzymatic assay (see below), except that reaction time was 1 min. All the values given are averages of at least duplicate assays. The enzymatic kinetic parameters were determined using the Grafit software (Leatherbarrow, 1990). 2.6. pH rate profiles Unbound enzyme (10 pg> and the equivalent amount of immobilized form were assayed at different pH, between 7.2 and 9.0. SL concentration was 2.5 mM (with Triton X-100 10 mM1. Conditions as described in Enzymatic assay. Optimum pH was mathematically calculated by mean expression.

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charged pancreatic PLA, (isoelectric point 6.3) was adsorbed preferentially to DEAE-Sephadex with high fixation level at pH 8.5 but with weak interaction at acidic medium. The high retention of bee venom PLA, activity upon interaction with the support (see below) is indicative that the catalytic domain involving His-48 and Asp-99 (Kuchler et al., 1989) would not participate in the binding process to CM-Sephadex. 3.2. Injluence of enzyme /support level

CM-Sephadex

DEAE-Sephadex Fig. 1. Immobilized

PLA,

from

&is

mellifera venom. Fixa-

Assay conditions: pH 8.0 for CM-Sephadex C-25 and pH 8.5 for DEAE-Sephadex A-25. Bars represent mean k SEM. tion level for Sephadex-type

supports.

3. Results and discussion 3.1. Enzyme immobilization

Under the conditions tested, practically all the enzyme present in solution was fixed to CM-Sephadex, reaching a fixation level of 15.3 mg g-‘. Fixation of Apis melfifera PLA, to the cation exchanger CM-Sephadex support was markedly superior to DEAEGSephadex (anion exchanger) as shown in Fig. 1. A better fixation level to DEAE-Sephadex was not possible even when the immobilization time was extended. The presence of several Lys and Arg residues in the sequence of PLA, from bee venom (Shipolini et al., 1974a) is indicative of its basicity, with an isoelectric point of 10.5 (Shipolini et al., 1971). The net positive charge of this PLA, at pH 8 suggests that the enzyme binds to the cation exchanger mainly by ionic interaction. However, van der Waals forces and hydrophobic interactions may also be present, since considerable interaction takes place even when both the enzyme and the matrix were positively charged. The importance of the electrostatic interaction of enzyme-support binding was reported in a previous work (Madoery and Camusso, 1991). In this report, the negatively

ratio on fixation

The extent of binding of enzymes to supports and the total amount bound will depend on the initial concentrations of catalyst and support, and the ratio of both components (Lilly et al., 1973). We found that the relationship between fixation and that ratio was nearly linear in the range 0.016 to 0.048 mg of enzyme per mg of support (see Fig. 2). This result suggests that enzymes have a great affinity for CM-Sephadex support. Under the conditions tested, the support accepted the virtually 100% of the enzyme originally in solution.

Initial ratio of enzyme/support

(mg mg-‘)

Fig. 2. Influence of initial enzyme/support ratio on fiiation level to CM-Sephadex C-25. PLA, (1.2 mg) and 50 mM Tris pH 8 (4 ml), in presence of: 75,50 and 25 mg of support. Bars represent mean * SEM.

R. Madoery et al. /Journal

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of Biotechnology

100 80

0 E B

149

ing to a more favourable spatial arrangement for the enzyme-substrate complex formation. In agreement with this is the fact that the retention activity of aminoacylase enzymes adsorbed on DEAE-Sephadex, decreased with an increased amount of immobilized protein (Lilly et al., 1973). Furthermore, a higher enzymatic load would contribute to an increased limitation of substrate diffusion and therefore a decreasing IE efficiency (Trevan, 1980).

t .* .$

40 (1995) 145-153

80 :

3.4. Retention of the activity and calcium requirement 1

01 10

15

20

25

Fixed enzyme to

30

35

40

45

50

support (mg gal)

Fig. 3. Bioconversion efficiency of PLA,-CM-Sephadex system. Influence of enzymatic loading on activity of immobilized systems with different fixation level (mg enzyme per g support). Enzymatic assay and PLA, activity measurement were performed according to Materials and methods. Substrate: SL 2.5 mM, 1 mg of IE. Bars represent mean f SEM.

3.3. Bioconversion efficiency of the IE systems

The effect of amount of immobilized PLA, on retention activity was studied. Results from experiments at three fixation levels are shown in Fig 3. A higher specific activity of bioconversion (efficiency) was achieved at the lowest fixation level tested. These results indicate that under conditions in which a high enzymatic load is obtained, only a fraction of enzyme would be involved in the catalytic reaction as has been described for other immobilized enzymatic systems (Trevan, 1980). A lower loaded system has a higher specific activity because the enzyme molecules would have less steric restrictions lead-

Immobilized PLA, maintained about 82% of its specific activity compared with the soluble form using SL/Triton mixed micelles as substrate and measured at 5 min (not initial rate, Table 1). In this condition about 35% of the substrate was hydrolyzed. The activity of soluble PLA, toward pure PC/Triton mixed micelles was 2.4-fold greater compared with total soybean phospholipids (SL), but retention activity remained almost unchanged (Table 1). A high retention activity suggests that the binding process takes place without substantial conformational changes of the protein. The native enzyme from bee venom has an absolute requirement of calcium (Shipolini et al., 1971). In a recent report, it has been described that calcium ion is involved in both processes: the binding of the enzyme to the substrate and in the catalytic coordination of the hydrolytic step. Two glycine residues and one aspartic acid &p-49) seem to be involved in the calcium binding (Kuchler et al., 1989). Our experience with the enzyme handling indicates that, at high substrate concentration (30 mM) the IE maintained about

Table 1 Normalized activity at 5 min (extent of hydrolysis) Substrate Total soybean lipid (SL) Soybean PC

CM-Sephadex (control)

PM2

Immobilized PLA, (retention activity)

0.00 0.00

1.00 2.40

0.82 2.10

Soluble

The activity was measured after 5 min using phospholipid/Triton X-100 1:4 (molar ratio) as substrate. The lipid concentration was 10 mM. The activity of the soluble enzyme was taken as 1.

R. Madoery et al. /Journal

of Biotechnology 40 (1995) 145-153

3.6. Operational and storage stability of IE system

20

0 7.5

8.0

8.5

9.0

PH

Fig. 4. pH-activity profiles for unbound (0) and immobilized PLA, (0). Conditions as Fig. 3. Bars represent mean+SEM. The activity at pH 8.3 for soluble and 8.2 for the immobilized enzyme were taken as 100%.

61% of its activity without

addition of calcium into the medium, taking the value obtained with 5 mM Cl,Ca as 100% of activity. However, the activity was null in the presence of 1 mM EDTA without calcium addition. These results indicate that traces of calcium coming with the substrate are enough to activate the system.

IE system was recycled several times to study its operational stability. After eight cycles, IE system maintained the initial activity level (see Fig. 5). After the first cycle the IE system is activated (20%) and in the successive cycles the activity falls to the initial value (Fig. 5). This effect was systematically observed in all the experiments performed. The physicochemical reason of this effect is unknown. Nevertheless, the activity remained almost constant after the second cycle indicating that enzyme is not desorbed from the support by partition into the substrate micelles. PLA 2 immobilized to CM-Sephadex conserved the total activity after 4 months of being stored at 5” C as dried enzyme-support system. 3.7. Effect of immobilization on enzyme activity: apparent kinetic parameters

The kinetic behaviour of both enzyme forms were comparatively studied. For soluble PLA, a K, of 12.9 mM with a V,,, of 883.4 pmol mgg’ min _ ’ (Fig. 6a), and for the immobilized PLA, K, 3.9 mM and a V,, of 306 pmol mgg’ min-’

3.5. pH dependence of enzymatic activity Fig. 4 shows the pH dependence profiles for both soluble and immobilized PLA, against SL/Triton mixed micelles. We found that the pH optimum was similar for both soluble (pH 8.3) and IE (pH 8.2); but the effective rate of PLA, became less sensitive to pH changes for the immobilized system. At least between pH 7.2 and 9.0 the activity of IE remains over 70% of its optimum, whereas the unbound form drops to below 45% at these extremes in the assayed conditions. It has been reported that immobilized enzyme molecules could be preserved from conformational alterations induced by pH changes by a protective effect of the support (Trevan, 1980).

0’ 0

1 12

1

1

3

4

5

6

7

8

9

Cycle number

Fig. 5. Operational through successive

stability of reuse assay. Enzyme activity cycles. Bars represent mean k SEM.

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600

of Biotechnology 40 (1995) 145-153

151

t c

0

20

0

60

40 Substrate (mM1

300

I

lb)

250

200

i

150

100

50

I

0

0

I 10

I

20

I

30

J

40

Substrate ImM)

Fig. 6. Kinetic parameters of PLA,. (a) Unbound PLA, activity toward SL/Triton X-100 mixed micelles (1:4 molar ratio); Vmax= 883.4 mmol mg-’ min-’ and KM = 12.9 mM. (b) CM-Sephadex PLA, activity against SL-Triton X-100 mixed micelles (I:4 molar ratio). 1 mg of IE with a fixation level to CM-Sephadex of 15.3 mg.g-‘. V,, = 306 mmol mg-’ min-’ and K, = 3.9 mM. The enzymatic activities were measured at initial rate (1 min). Bars represent mean f SEM.

152

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of Biotechnology 40 (1995) 145-153

(Fig. 6b). Double-reciprocal plots represented in the insert of Fig. 6 are linear for both enzyme forms, but for IE this is true only up to 30 mM of SL substrate. Soluble PLA, adsorbed at lipid surfaces acts in the scooting mode (Jain and Gelb, 1991). In the scooting kinetic mechanism the enzyme remains at the surface of the lipid particle several catalytic cycles without desorption. This condition is hard to fill for immobilized PLA, if the enzyme interacts strongly with the support. Theoretically, a lower apparent K, obtained for the IE system indicates a higher affinity for substrate, and it is interpreted as a higher superficial affinity of the enzyme for the lipid micelles. However, the immobilized PLA, has some restriction for a strict scooting mechanism leading to a lower activity and V,,,. The kinetic parameters of enzymes bound to a support are often affected by the immobilization process. This may be due to conformational changes, steric effects, diffusional limitations or partitional effects, conducting to deviations of the Michaelian behaviour. In our case, the slopes from double-reciprocal plots were similar, the apparent K, for the IE was lower and the linear relationship of l/v vs. l/S was up to 30 mM of SL. This indicates that there are no strong diffusional limitations of the aggregated substrate, at least in the working range of the substrate concentration tested. A classical interpretation of the data would be that the immobilization system works as an anticompetitive inhibition effect upon enzyme-support interaction (Segel, 19751, probably due to a loosening of the typical scooting mode of enzymatic hydrolysis of phospholipases. However, future kinetic investigations should be performed to elucidate the mechanism of action of immobilized PLA,.

4. Conclusions Immobilization assays showed that PLA, from bee venom has strong affinity for CM-Sephadex and the interactions seem to be mainly electrostatic. A high operational stability was found, indicating that enzyme-support bound was stable in reusing assay conditions. This aspect may be of

great importance for applications in continuous biotechnological process. Also, the high retention activity of the immobilized system indicates that negative effects on protein conformation are not present. As a consequence of immobilization, the PLA, was less sensitive to pH changes and it had an excellent storage stability. Under the assay conditions investigated we concluded that there are no strong diffusional limitations or partitioning effects to promote deviations on Michaelian behaviour. The investigation developed in the present work would give the basic information for obtaining an immobilized system with high potential application for continuous phospholipid bioconversion.

Acknowledgements

This work was supported by SECyT UNC, CONICOR and CONICET, Argentina. GDF is career researcher from CONICET. The authors are grateful to Dr. I.D. Bianco for critical reading of the manuscript.

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