Immobilisation/stabilisation on different hydroxilic supports of lipase from Candida rugosa

Journal of Molecular Catalysis, 83 (1993) 261-271 Elsevier Science Publishers B.V., Amsterdam

261

MO146

Immobilisation/stabilisation on different hydroxilic supports of lipase from Candida rugosa

Miguel Arroyo, Jose Ma. Moreno and Jose V. Sinisterra* Organic and Pharmaceutical Chemistry Department, Faculty of Pharmacy, Universidad Complutense, 28040 Madrid (Spain); tel. (+34-1)3941823, fax. (+34-1)3941822

(Received October 8,1992; accepted February 16,1993)

Abstract

Lipase from Candida rugosa has been covalently immobilized on tosyl activated matrix (agarose and corn cob) and optimum immobilisation conditions determined. The immobilized derivatives exhibited greater residual activity than the ones reported previously. Studies on the activity and stability of the different insolubilixed derivatives prepared showed that the enzymatic derivative immobilized on small corn cob is resistant to inactivation by temperature, at 50°C. This derivative was 100 times more stable than its soluble counterpart. The insolubilized derivatives are more active and stable at higher temperatures ( > 35 ’ C ) than soluble enzyme. Lipase activity (using olive oil emulsion) and e&erase activity (usingp-nitrophenyl butyrate) have been determined. Kinetic studies have been carried out with soluble and immobilized derivatives. The influence of Na (I) and Ca (II) on the lipase and e&erase activities is discussed.

Key words: Candida rugosa, enzyme immobilization, lipase

Introduction

Due to the high regio- and stereoselectivity of lipases, besides their high catalytic activity in organic solvents [ 11, they have received growing attention in organic synthesis. Lipase from Candida rugosa is an interesting enzyme with high activity in hydrolysis as well as in synthesis; and it discriminates between different substrates [ 21. Therefore, the immobilisation of lipase can offer several advantages, for both industrial and clinical applications [3], including repeated use, easy separation of reaction products from the biocatalyst, im- provement of enzyme stability, and continuous operation in fixed packed-bed reactor.

It is generally accepted that enzyme-support multipoint attachment may exert an important stabilizing effect on the insolubilized enzyme derivatives [ 41. However, strategies for the preparation of lipases linked to many supports by adsorption of hydrophobic bonds have not been very successful, especially when these derivatives have been used in organic synthesis. In general, the

*Corresponding author.

0304~5102/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved.

262 M. Arroyo et al. / J. Mol. Catal. 83 (1993) 261-271

activities of the immobilized lipases so far reported are considerably lower when they are compared to those displayed by other immobilized enzymes. Lipolytic activities between 1-2% (with native enzyme) have been reported for immobilized lipase on Biogel [5], and Spherosil beads [6]. Recently Shaw et al. [7 ] and Otero eta/. [ 8 ] have reported higher activities (10% or 30%, respectively). Kosugi et al. [9] suggested that the ionic and hydrophobic interactions between the lipase and the surface of the support were important for the non- covalent immobilisation of lipase. Based on this concept, Yamane et al. [10] used octyl- and phenyl-sepharose CL-4B to immobilize lipase obtained from Candida. When tested in oil hydrolysis, the lipolytic activity of this immobilized lipase decreased gradually because the lipases were only loosely attached to the support.

In a previous paper we have proposed an adequate immobilisation/stabilisation procedure of enzymes to tosyl activated supports. This methodology is a modification of the one described by Mosbach et al. [11]. The attachment of enzymes proceeds through their amino groups to monolayers of tosyl groups built on the hydroxyl groups of agarose gels [12] and corn cob [13]. This approach, which has important advantages in the achievement of enzyme- support multipoint attachments, has been successfully applied to the immo- bilisationofBenzonase [ 14 ],StaphylococcalNuclease [ 15 ] andPancreaticRibo- nuclease [16].

In the present paper we show the application of this methodology to the immobilisation/stabilisation of C. rugosa lipase. A complete analysis of the variables that control the insolubilisation process is presented.

M a t e r i a l s a n d m e t h o d s

Lipase from Candida rugosa (E.C. 3.1.1.3) (type VII, containing 1010 U/ mg solid using olive oil) and lipase substrate (stabilized olive oil emulsion 50 vol.-% or p-nitrophenyl butyrate) were obtained from Sigma Chemicals.

Corn cob particles (EU-GRITS, 0.84-0.54 and 4.0-2.0 mm particle size) were kindly provided by Bio-Europe, Tolouse (France). Agarose gel beads (Bio- gel A-150 m, 100-200 mesh) were from Bio-Rad Laboratories (Richmond, USA); and 4-toluenesulfonyl chloride was from Merck. All other chemicals were of reagent grade.

Storage of native enzyme A weighed amount of the powdered commercial enzyme was dissolved at

4 ° C in 0.1 M Tris/HC1 buffer, pH = 8.0 (standard buffer) and left with gentle stirring. After 2 h, the mixture was centrifuged at 5000 rpm for 15 rain and the insoluble material rejected. This enzyme solution, when not used immediately, was stored at 2 o C.

M. Arroyo et al. /J. Mol. Catal. 83 (1993) 261-271 263

Immobilisation process The activation of agarose and corn cob was carried out according to the

tosylation method described previously [ 12,131. The immobilisation of lipase was carried out at 4”C, for 3 h. with slow stirring in the standard buffer (0.1 M Tris/HCl buffer, pH= 8.0). One gram of each support was added to the enzyme solutions, with different concentrations. 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 and the activity of the filtrate of immobilisation process, (E)im. The Lowry method [ 171 cannot be used because the tosyl groups react with the reagents.

The amount of enzyme adsorbed on the support, (Ejads, was measured by contacting the immobilized derivatives with phosphate/NaOH buffer (pH=7.5) solution with slow stirring overnight at 2°C. After this time, the activity was measured to determined the amount of adsorbed enzyme (Table 2). The covalently bonded enzyme (E),, was determined by the difference (Elim- (E)acl*

Enzyme assays The hydrolysis of olive oil emulsion (lipase activity) was carried out in 1

mM sodium phosphate/NaOH buffer (pH=7.5) at 35°C. The acid released was continuously titrated to pH = 7.5 with the aid of a pHstat Crison Model MicroTT 2022. Several NaOH solutions (1 to 10 mM) were used as titrating agents. The agitator speed was 50% and the total reactor volume was 5 ml.

The e&erase activity was measured in a spectrophotometer UV-visible Shimadzu model UV-2100 (with magnetic stirring in the cuvettes), by the increase in the absorbance at 346 nm according to the method of Fletcher et al. [ 18 1. In this case, we carried out the hydrolysis of ap-nitrophenyl butyrate in 0.1 M Tris/HCl buffer (pH = 7.0) at 35 ‘C. The corn cob derivatives float on the liquid and the agarose does not absorb in the UV zone. There is thus no interference by the support in the assays. Mass transport limitations were not observed under the experimental conditions used.

The catalytic efficiency of immobilized derivatives, was determined - using initial rates - as the ratio between the specific enzymatic activities of 3 mg of native enzyme and the amount of immobilized derivative which contains 3 mg of enzyme, only taking into account the covalently bonded enzyme molecules (El,,, (Tables 1 and 2). All of the data are the average of triplicate samples and are reproducible within 2 5%.

Results and discussion

Immobilisation of the enzyme on agarose The results of the immobilisation process of C. rugosa lipase on agarose,

previously activated with 4-tosyl chloride are shown in Table 1. The percentage

264

TABLE 1

M. Arroyo et al. /J. Mol. Catal. 83 (1993) 261-271

Results of the immobilisation of lipase on agarose’

Entry PH Lipase added (mg)/ml wet gel

(W,, (%)

Catalyticb efficiency ( % )

A-l A-2 A-3 A-4 A-5 A-6 A-7 A-8

25 12 a2 25 29 76 25 43 24 37.5 32 75 50 45 70 62.5 52 75 75 40 73

100 35 76

*Activation degree = 8.8 pmol tosyl/g support (3.4 tosyl groups/l0 nm2). bLipase activity using olive oil emulsion.

of adsorbed enzyme (EJads was between 0.9-3.5% on this support, (E),,, increases with pH (entries A-l to A-3), as may be expected from the basic prop- erties of the e-NH, group of lysine. Nevertheless, the residual activity decreases with pH, as expected from the pH profile of C. rugosa lipase. When the amount of lipase added to the immobilization mixture increases, the percentage of immobilized enzyme increases up to 52% (entries A-2 and A-4 to A-8). The percentage of immobilisation obtained by our method is similar to or greater than that described by Otero et aZ. using the glycidol method [8] or by other workers [ 5-71. The maximum amount of enzyme bonded to the activated agarose (entries A-6 to A-8) was 30-35 mg enzyme/ml wet gel. This amount can be considered as the maximum enzyme load of the activated support.

The percentages of immobilized enzyme were similar to those reported by Lavayre and Barati [6] using spherosil as support. On the other hand, the percentage of protein bonded per gram of support (E),,, achieved in the present study is lower than the value reported by Otero et al. (89-100% ) [ 81. The high percentages obtained by these workers may be related to the small amount of enzyme added to the sepharose in the immobilisation process (12.5 mg enz/ g wet gel, lower than the amount we used (Table 1) ) and with the high degree of activation of the supports (18 aldehyde groups/l0 nm2 [ 81) in comparison with ours (3.4 tosyl groups/l0 nm2). This allows us to assume a multipoint attachment in the first case, and monobonding in our case. The enzyme load is higher than the value of 0.5 mg enz/ml gel of Sepharose 4B reported by Kilara et al. [ 191 or Shaw et al. [9] (19 mg/ml gel using chitosan as support) or Carta et al. [ 201 (10 mg/ml gel using Nylon 6 as support).

Immobilisation of the enzyme on corn cob Corn cob is an excellent cellulosic support for the immobilisation of nu-

cleases [ 13,14,21]. Two different particle size were tested to analyze the diffusional problems. The results obtained are shown in Table 2.

M. Arroyo et al. /J. Mol. Catal. 83 (1993) 261-271

TABLE 2

265

Results of the immobilisation process on corn cob

support Entry Lipase added

(mg) /g corn cob

(E& (%)

(E,,)* Catalytic efficiency’ ( % )

Corn cob’ (0.5-0.8 mm0)

C-l 62.5 4.79 20.13 33.0 c-2 125 8.58 29.22 25.3 c-3 250 12.31 51.69 2.3 c-4 375 16.09 63.51 1.1 c-5 500 16.53 50.08 0.6

Corn Cobb (2-4.0 mm0)

C-6 50 21.00 28.10 16.2 c-7 62.5 20.53 42.37 17.8 C-8 125 20.26 47.74 16.5 c-9 250 27.90 53.90 9.9 c-10 375 30.80 49.10 4.5

“Activation degree=32.5 pmol tosyl/g support (25.23 tosyl groups/l0 nm2). bActivation degree= 15.6 pmol tosyl/g support (12.87 tosyl groups/l0 nm2). “Referred to (E),,. *Referred to the amount of native lipase added in the immobilisation process.

Two different enzyme molecules are present in these derivatives. The weakly adsorbed enzyme molecules, (EJads, which can be removed after stirring overnight at 2°C (longer stirring times do not remove more enzyme molecules), and the covalently bonded enzyme molecules (E),,,. These values are expressed as percentages of the amount of native enzyme added in the immobilization process. When the amount of added enzyme increases, (E),,, increases, but the catalytic efficiency decreases. This behaviour is different than that observed with agarose (Table 1) , where the catalytic efficiency remained constant. This decrease of residual activity seems to be related to a multilayer interaction of the enzyme on the corn cob support due to the low surface area. Only the internal layer can actually be covalently bonded to the support by the e-NH, groups of the protein or by the -CH,OH groups of the sugar residues of the glycoprotein:

n OTs + HgN-enzyme - n HN-enzyme + HOTS

n OTs + HOCH, -sugar - n -OCH, -sugar + HOTS

The upper enzyme layers could be bonded by hydrophobic and/or hydro- gen bonds to the covalently bonded enzyme molecules through a protein chain or sugars which appear in the lipase structure (C. rugosa produces two kinds of extracellular lipase, lipase I of M, 60,000 as a major component and lipase II of M, 60,500 as a minor one, both showing 7% glycosylation) [22]. These kinds of bonds are more energetic than those of physical adsorption and so these enzyme molecules cannot be removed under the storage conditions which we used (2 o C ) . Nevertheless, these enzyme molecules are quickly deactivated

266 M. Arroyo et al. 1 J. Mol. Catal. 83 (1993) 261-271

under the reaction conditions (35 o C ) (Fig. 1). Thus we could say that are two different kinds of strongly bonded enzyme molecules in this derivative: (i) true covalently bonded, and (ii) multilayer H-bonded enzyme molecules. The reduction in the catalytic efficiency would be greater with large amounts of multilayer bonded enzyme molecules than with low percentages of adsorbed enzyme, as is observed in Table 2 for C-3, C-5, C-9, and C-10 derivatives.

The particle diameter of the corn cob has no great influence on the maximum value of (IQ,,, (Table 2 ) . However, the catalytic efficiency is lower in the large-grained corn cob (2-4 mm diameter) than in the small-grained one (0.5-0.8 mm diameter) (16.2% against 33%, entries C-6 and C-l, respectively, in Table 2 ) . This finding can be explained because in the small corn cob (with a similar amount of bonded enzyme: 14 mg enz/g support in C-6 and 12.5 mg enz/g support in C-l) the number of multilayers is lower than the derivative immobilized on the great particle size corn cob due to its higher surface area value. Thus, the greater the percentage of multilayer enzyme molecules present, the greater is the deactivation rate and the lower the catalytic efficiency.

Thermal stability The immobilized derivatives can be stored at 2 “C for one month, without

any appreciable loss of catalytic activity. The storage stability of native and insolubilized lipase has been studied at 50 ’ C, representing extreme conditions (Fig. 1). The plots of residual activity in relation to the storage time adjust

20 30

Time (hours)

Fig. 1. Comparative deactivation of three different derivatives and soluble enzyme stored at 50” C. Lipasic activity, pH~7.5, T=35”C, [substrate] =2.275 mg/ml, VT=5 ml. (Symbols and amounts

added to reactor: 0 native enzyme; 0 C-10; 0 A-4; A C-2.)


quite well to a double exponential decay (with the exception of the soluble enzyme, which is only adjusted to a single exponential decay). In all cases, the exponent of the first decay was greater than the second one </?J/3Z x 100). Us- ing the first exponential decay we have calculated the half-life of the derivatives, the stabilisation factor (referred to the half-life of the native enzyme), and the initial deactivation rate constant, p1 (Table 3).

We can observe that the insolubilized derivatives in agarose A-2 and A-4 (Table 3) are most resistant to the thermal deactivation. These derivatives were prepared with low enzyme concentrations (25 and 37 mg enz/ml wet gel). in order to achieve a maximum amount of true covalently bonded enzyme (E),,. The other derivatives show high values of a, as may be expected from a multilayer interaction. The half-life achieved by our method (26-27 h) is lower than that achieved by the glycidol-agarose method (60-70 h [ 8 J ), probably due to the lower number of enzyme-support fixation points in our method than in the other.

In the case of corn cob (Table 3), the immobilized derivatives obtained with the small particle size corn cob (entries C-2 and C-4) give higher half-life values than those obtained with a large particle size (entries C-8 to C-lo), where the multilayer interaction is very high.

In the case of corn cob derivatives, /3 depends on particle size (Table 3 ) . We observe that the derivatives obtained on the small particle size corn cob display lower B values (C-l to C-4) than those obtained on a large particle size support (C-8 to C-10). Therefore, the deactivation rate constant depends on the nature of the support and, for one kind of support, depends on the particle

TABLE 3

Thermal stability of native and immobilized C. mgosa lipase

Derivative Half-life Stabilisation Deactivation

type (h) factor” rateb/3 (h-l)

Soluble 0.5 1 1.305 A-2 26 52 0.119 A-4 27 54 0.114 A-5 18 36 0.137 A-6 18 36 0.136 A-7 16 32 0.147 A-8 16 32 0.148 C-l 42 84 0.031 c-2 50 100 0.024 c-4 45 90 0.065 C-8 4 8 0.204 c-9 4 8 0.170 c-10 6 12 0.111

“Relation between soluble and derivatives half-lives. bSingle exponential decay equation: Y = A,e -@+ A2

268 M. Arroyo et al. /J. Mol. Catal. 83 (1993) 261-271

size. Similar conclusions have been obtained when the enzyme is immobilized on polymers [ 71.

Influence of pH and temperature on enzyme activity The enzymatic activity of the native and immobilized derivatives has been

tested at different pH and temperatures. This study has been carried out in presence of Ca( II) (3 mM) and Na (I) (200 mM) as described in the literature [23]. In all cases, the optimum temperature was 35”C, yet the immobilized enzymes are more stable than native enzyme (Fig. 1) .

Kilara et al. [ 191 and Shaw et al. [ 71 have reported that the immobilization of lipase on Sepharose 4B, PVC, Chitin and agarose caused the optimum reaction pH to shift from 7.5 to 8.5. We also found that the optimum reaction pH of lipase shifts from 7 to 8 after immobilizing the enzyme on agarose (A- 4) and the large-grained corn cob (C-10) (Fig. 2). The small-grain corn cob derivative (C-5) displays the same behaviour as the native enzyme (optimum pH = 7.5). The hydrophilic nature of the support apparently has little effect on the extent of the pH shift, probably because both supports present polysac- charide structures.

Kinetic parameters Lipase activities (measured with olive oil emulsion) and e&erase activi-

ties (measured withp-nitrophenyl butyrate) have been determined for soluble and immobilized derivatives. The lz,, and K, values were determined from

Activity 1%)

6 6.5 7 7.5 8 8.5 9

PH

Fig. 2. Influence of pH on the enzymatic activity of soluble and immobilized derivatives. Lipasic activity, T= 35 ’ C [substrate] = 2.275 mg/ml, V, - - 5 ml. (Symbols and amounts added to reactor: 0 native enzyme; 0 C-10; A C-5; 0 A-4.)


kinetic data using the Lineweaver-Burk equation. The results are shown in Table 4.

The lipase and esterase kcat values obtained for the immobilized enzymes in the presence or absence of Na (I) and Ca (II), respectively, are lower than for the native enzyme, as can be expected from the catalytic efficiency values (Tables 1 and 2). We observe that the specific rate constant, k,,,, increases in the presence of Na(1) and Ca( II) ions, in the case of lipase activity (entries soluble vs. soluble’, or A-7 vs. A-7’ in Table 4). This effect is well documented in the literature [9,24-261; Na(1) favours the emulsification of lipids, giving. micelles [24], increasing the interfacial lipid-water surface; and Ca(I1) removes the fatty acids produced during the hydrolysis [ 261. Both effects increase the hydrolysis of lipids by lipases. However, the e&erase kcat constant decreases in the presence of ions in the case of the native enzyme (entries soluble” vs. soluble”‘), while insolubilized derivatives showed similar kcat values in the presence and absence of ions (entries A-7” vs. A-7”‘, or C-lo1 vs. C- 10” in Table 4). Similar effects have been reported by Rhee et al. [ 27,281 with C. rugosa lipase-catalyzed hydrolysis of olive oil emulsion in a reverse-phase system.

From these results we conclude that the presence of cations is very important in the case of lipase activity that takes place in the water-oil interface, but not when the hydrolysis takes place in the homogeneous phase (e&erase activity). This fact must be related to the stabilisation of the micelle structure by Na(1) and to the interface cleaning effect of Ca(II), which removes the reaction products from the interface.

TABLE 4

Influence of immobilisation process on kinetic constants

Derivative

We

Ions” Activityb K,,, (mg subs./ml)

k cat (pm01 subs./ml min mg enz.)

Soluble Soluble’ Soluble” Soluble”’ A-7 A-7’ A-7]’ A-7”

c-9 C-9’ C-10’ C-10” C-10”

No Yes No Yes No Yes No Yes Yes No Yes No Yes

L 0.375 1.45 L 2.395 72.36 E 0.058 272.02 E 0.032 115.04 L 0.876 4.91 L 0.770 16.68 E 0.063 43.30 E 0.035 47.14 L 1.314 1.38 L 4.630 0.91 L 0.670 1.45 E 0.081 1.05 E 0.079 0.80

“200 mM Na(1) and3 mM Ca(I1). bL = lipase activity; E = e&erase activity.

270 M. Arroyo et al. / J. Mol. Catal. 83 (1993) 261-271

In the hydrolysis of olive oil, K,, which is related to the maximum packing density of the substrate, is mainly increased by the presence of Na (I) in the case of the soluble enzyme, but decreases in the case of insolubilized derivatives. On the other hand, K, diminishes in the e&erase activity of soluble and immobilized derivatives.

In the hydrolysis of olive oil, the presence of ions affects the K, value in different ways. While a large increase in K, value is observed in the case of the native enzyme (entries soluble vs. soluble’), a strong reduction is observed for the corn cob derivatives (C-9 vs. C-9’). This finding is evidently related to the formation of interfaces, which improves the enzymatic activity of native enzymes due to the presence of ions. The increase in the ionic strength removes the multilayer enzyme molecules in the weak derivative obtained from corn cob, giving very poor results. In the case of agarose (entries A-7 vs. A-7’), where all the enzyme molecules are practically covalently bonded in mono- layer, the effect of the presence of ions is negligible because the geometry of the interface is controlled by the structure of agarose.

Finally, we compare the effect of immobilisation on esterase and lipase activities, and we see that soluble lipase has a ratio ApNPB/Aol+e oil of 190 in the absence of ions, while this relation decreases upon immobilisation (i.e., the corresponding value for derivative A-7 was 10). This effect has been reported in the literature [ 81, and is related to diffusional and interfacial problems.

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Immobilisation/stabilisation on different hydroxilic supports of lipase from Candida rugosa