BIOTECHN’OLffiY TEtZKWmS Volume 9 No. 1 (Amary 1995) p. l-6 R waived 7th Norm her

Reducing enzyme conformational flexibility by multi-point covzilent

immobilisation

R. Fernandez-Lafuente*, A.N.P. Wood & D.A. Cowan.

Department of Biochemistry and Molecular Biology

University College London

United Kingdom.

A thermostable esterase was generate “limited-linkage”

immobilised to glyoxyl-agarose under conditions designed to and “multi-uoint” covalent derivatives. The multi-noint

derivative was 830-fold more thermostab& than the limited-linkage derivative and &.&ed more activity in the presence of sodium chloride and organic solvents. Medium chain (C8) aliphatic p-nitrophenyl ester substrates, which inactivate the soluble enzyme, were shown to be more readily hydrolysed. Together these data support the contention that multi-point covalent immobilisation results in a more rigid, less conformationally flexible protein structure.

SUMMARY

INTRODUCTION

Multi-point covalent linkage of proteins to glyoxyl-agarose gels has been described as a

technique which increases enzyme rigidity and thus should enhance enzyme stability with

respect to denaturing agents (e.g., temperature, extremes of pH, organic solvents and

urea) [Guisan et al., 19911.

Where enzyme rigidity has been increased by multi-point covalent immobilisation, this

should be reflected in a higher resistance to changes in reaction conditions; i.e., the

enzyme might be less “sensitive” by being partially “frozen” via the multi-point covalent

interactions with the support.

Using well establish methodologies for generating multi-point and limited-linkage

covalent enzyme-glyoxyl agaroses [Guisan, 19881, we have prepared different derivatives

of a thermophilic esterase from Bacillus stearothermphilus [Fernandez-Lafuente et al., in press]. The behaviour of these derivatives in the presence of solvents and under non-

native conditions is presented.

MATERIALS AND METHODS

Glyoxyl CL 10% agarose B gels, prepared as previously described [Guisan, 19881, were generously donated by Hispanagar S.A. (Fax No. 34-47-20 03 28, Burgos, Spain). All reagents were from Sigma Chemical Co., Poole, Dorset, UK. Semi-purified esterase extracts were obtained from Bacillus stearothermophilus strain Tokl9Al as previously described wood et al., in press].

The limited-linkage esterase derivative was prepared as previously described [Fernandez- Lafuente et al., in press]. The multi-point esterase derivative was prepared using a similar protocol, but with a 30h incubation period at 3OOC.

Esterase activity was assayed spectrophotometrically by monitoring the increase in absorbance at 405nm during the hydrolysis of p-nitrophenyl esters. Assays were performed in a lcm path length cuvette provided with magnetic stirring and thermostatically controlled to 30°C. The standard assay used 2mL of 50mM sodium phosphate, pH 7.0 and 15l.tL of 1OOmM p-nitrophenylpropionate (p-NPP). Activity in the presence of solvents and salts was determined by inclusion the additives in the reaction mixture at the concentrations specified.

Irreversible enzyme inactivation [Guisan et al., 19921 was determined by incubation of the derivatives at 72OC in O.lM phosphate buffer, pH 7.0 and assaying residual activity under standard conditions.

RESULTS

Preparation of multi-point covalent esterase derivative

Figure 1 shows the time-course of esterase activity during the preparation of an esterase-

agarose derivative using reaction conditions designed to generate a multi-point enzyme-

support interaction. This derivative was used for subsequent characterisation. Under the

preparation conditions (30°C, pH lO.O), the “blank” suspension (i.e., esterase incubated

with chemically reduced glyoxyl-agarose) lost 20% of the initial activity over the 30h

incubation period. This implies that approximately 30% of the activity lost during

Figure 1. Time-course of activity

during preparation of multi-point

covalent esterase derivatives.

[Open circles, immobilisation

suspension; Closed circles, “blank

suspension”; Closed squares,

supernatant of the immobilisation

0 suspension] 0 5 10 15 20 25 30

TlME (hours)

2

preparation of the immobilised derivative (60% of initial activity in 30h) might be

attributed to denaturation and/or inactivation of the enzyme. The additional loss of activity

during the immobilisation process will be derived from processes involved in the enzyme-

support interaction. These may include unfavourable enzyme-matrix interactions which

may result from high covalent linkage densities.

The generation of a multi-point covalent enzyme-support derivative was confirmed by the

high level of thermostabilisation when this preparation was compared with a limited-

linkage derivative (Figure 2). A comparison of the half-lives of denaturation indicated that

the multi-point derivative was approximately 830-fold more stable than the limited-linkage

derivative. Enhanced stability with respect to temperature and potential denaturants

(solvents, urea, etc) after multi-point immobilision of mesophilic enzymes has been

previously reported (Guisan et al., 1991).

100

80

60

40

20

0

Figure 2. Thermal inactivation of

limited-linkage (closed circles) and

multi-point (opened circles)

derivatives.

0 10 20 30 40 50 60

TME (hours)

Esterase activity in the presence of alcohols

It has been previously demonstrated that the presence of low concentrations of linear

aliphatic alcohols in esterase assays with p-NF’ esters resulted in significant increases in

the rate p-NP release [Wood et al., manuscript submitted to Biotech. Appl. Biochem.].

This effect was correlated with enhanced rates of transesterification. At higher

concentration, alcohols were also shown to decrease esterase activity, an effect partially

attributed to competitive inhibition.

Because of the potential application of this esterase in transesteritication reactions, activity

was monitored in increasing concentrations of two aliphatic alcohols, methanol and n-

propanol.

It is evident from Figure 3 that the activity of both immobilised-esterase derivatives

increased in the presence of methanol at concentrations of up to 20%. At concentrations

under 20% (v/v), this alcohol has been shown to stabilise the soluble esterase [Wood et

aZ., manuscript submitted to Biotech. Appl. Biochem.]. However, under similar reaction

conditions, n-propanol induced a significant reduction in the activity of the limited-linkage

derivative, while the more stable multi-point derivative retained higher levels of activity.

The significant reduction of esterase activity induced by n-propanol at concentrations of

greater than 6% v/v can be attributed in part to solvent-induced denaturation (n-propanol

at 9% v/v significantly enhances the denaturation rate of immobilised esterase:

unpublished data). However, direct active site effects (e.g., competitive inhibition) might

also be implicated in the reduction of esterase activity at high n-propanol concentrations.

The relatively high resistance of the multi-point derivative might imply a conformational

restraint to solvent-induced denaturation.

300

g 250

i 200

E 4 150

200

iz c 175

[ 150

< 125

0 5 10 15 20 0.0 2.5 5.0 7.5 10.0

pfmHANOL] % [PROPANOL] %

Figure 3. Effect of methanol [A] and n-propanol [B] on the activity of limited-linkage

(closed circles) and multi-point (open circles) immobilised esterase derivatives.

Activity in the presence of NaCl

Immobilised esterase derivatives assayed in the presence of NaCl (Figure 4) showed a

significant and salt concentration-dependent reduction of activity. Assayed under identical

conditions, the activity of the limited-linkage derivative was significantly more sensitive to

the presence of NaCl than that of the multi-point derivative. This result would be

consistent with a reduction of observed activity via salt-induced distortion of the enzyme

structure, where the different sensitivities of the immobilised derivatives reflect their

relative levels of molecular rigidity.

b Figure 4.

0 50 -

Eg 25-

@ 0 I I I

0 loo 200 300 400

WaCl] mM

Enzyme specificity

Activity of limited linkage (closed circles) and multi- point (opened circles) esterase derivatives in the presence of NaCl.

A comparison of the relative activities of limited-linkage and multi-point immobilised esterase derivatives on C2 to C8 p-nitrophenol ester substrates (Figure 5) suggests that the substrate specificity of the two derivatives is not identical. For example, where activities of the two derivatives are normalised (by choosing activity on pNP-propionate as lOO%), the ratios of C3/C8 activities are 5 and 2.5, respectively. Increased molecular rigidity in the multi-point derivative might be expected to disfavour the binding and catalysis of substrates which differ structurally from the C4 and C5 p-NP-esters (Figure 5), giving the reverse of the result observed. However, this behaviour may be explained in terms of the destabilising effect of the long chain (C8) ester [Wood et al., manuscript submitted to Biotech. Appl. Biochem.] where the retention of activity in the presence of this substrate favours the more rigid multi-point immobilised enzyme.

150

s E 120 -

6 901

i 604

Figure 5. Relative activity of limited-linkage (closed circles) and multi-point (open circles) esterase derivatives. [Substrates were p-

3 NP-acetate (C2) to p-NP-caprylate

2 30- (C8); Activity on p-NP-propionate

0 I I I I I is defined as lOO%] 2 3 4 5 6 7 8

ACYL CHAIN LENGTH

5

CONCLUSIONS

Multi-point covalent immobilisation of a thermostable esterase to glyoxyl-agarose matrices

generated a stabilised derivative with high resistance to thermoinactivation, miscible

hydrophilic organic co-solvents and high salt concentrations. The data supported the

hypothesis that multi-point immobilisation enhances the overall conformational rigidity of

the enzyme structure. This conclusion was also supported by apparent differences in

substrate specificity between the multi-point and limited-linkage immobilised enzymes.

These results suggest the multi-point immobilised enzymes may have characteristics

which are particularly suitable for biotransformation processes in non-native conditions.

ACKNOWLEDGEMENTS

The authors wish to thank Hispanagar S.A. (Burgos, Spain) for the kind gift of the

agarose gels. We also wish to thank Mr Paul Shadbolt (Helix Biotechnology Ltd, London

UK) for his excellent technical support. This work has been supported by a fellowship

from “Consejo Superior de Investigaciones Cientificas” (Spain) and by a Human Capital

and Mobility grant from the European Community.

REFERENCES

Fernandez-Lafuente, R., Cowan, D.A. & Wood, A.N.P. Enzyme Microb. Technol. In

press.

Guisan, J.M. (1988) Enzyme Microb. Technol. 10, 375-382.

Guisan, J.M, Bastida, A., Cuesta, C., Fernandez-Lafuente, R. & Rose& C.M. (1991)

Biotechnol. Bioeng. 30, 1144-l 152.

Guisan, J.M., Bastida, A., Blanco, R.M., Cuesta, C., Rodriguez, V. & Fernandez-

Lafuente, R. (1992) Biocatalysis in non-conventional media. (ed., J. Tramper et

al.) Elsevier SC. Publ., 221-228.

Wood, A.N.P., Fernandez-Lafuente, R & Cowan, D.A. Enzyme Microb. Technol. Zn

press.