B I O T E C H N O L O G Y L E T T E R S Volume 17 No.6 (June 1995) pp.603-608 Received 28th April

EFFECT OF WATER-MISCIBLE APROTIC SOLVENTS ON KYOTORPHIN SYNTHESIS CATALYZED BY IMMOBILIZED ¢z-CHYMOTRYPSIN

Pedro Lozano, Teresa Diego and Jose L. Iborra"

Departamento de Bioquimica y Biologia Molecular B e Inmunoiogia. Facultad de Quimica. Universidad de Murcia. P.O. Box. 4021. E-30001. MURCIA. Spain.

SUMMARY Immobilized a-chymotrypsin was used as catalyst to synthesize a kyotorphin derivative (Bz-Tyr-Arg-OEt) in the presence of five water-miscible aprotic solvents (dimethylsulphoxide, dimethylformamide, acetonitrile, acetone and tetrahydrofurane) at 30 °C. By using a kinetically- controlled approach, the maximum synthetic activity was obtained when Arg-OEt was used as nucleophile donor at a concentration 1.5-times higher than the acyl-acceptor substrate (Bz-Tyr- OEt). The water-miscible aprotic solvents enhanced greatly the synthetic activity proportionally to their hidrophilicity properties adequately measured by the log P parameter. At the optimum solvent concentration for the enzymatic peptide synthesis, both the water activity (Aw) of the media and the water content of the immobilized derivative showed a saturation profile against the log P parameter. As a function of the solvent hydrophilicity, these water parameters were shown as key parameters for the increase in the synthetic activity of the enzyme by the presence of these solvents.

INTRODUCTION

The use of biocatalysts in organic solvent has attracted much interest in last decade to make up

synthetic process by using hydrolytic enzymes. For protease-mediated peptide synthesis, the main

advantage in using organic solvents is the shift of reaction equilibrium to the synthetic way (Kise et

a1.,1988, Lozano et al.,1992, Cerovsky and Jakubke, 1994). However, the influence of water-

miscible organic solvents on enzymatic reactions are not straightforward. Organic solvents may act

as competitive inhibitor of enzymes, or by reducing the hydrophobic interactions between

substrates and enzymes or by changing the electrostatic interactions of polar groups in the protein

structure (Nagashima et a1.,1992). The consequence of conformational changes in the enzyme

structure and enzyme-substrate interactions by direct contact with organic solvents would be

changes in activity and specificity of the enzyme (Mozhaev et al., 1989, Lozano et al., 1993, 1994).

In this way, this paper describes the influence of five different water-miscible aprotic solvents

(dimethylsulphoxide, dimethylformamide, acetonitrile, acetone, and tetrahydrofurane) on the ct-

chymotrypsin activity to synthesize the analgesic dipeptide kyotorphin by kinetic control strategy

from activated substrates. The overall effect of these solvents was analyzed as a function of the

603

changes in the water properties, such as, water activity (Aw) of the reaction media and the water

content of the immobilized derivative, as well as, the hydrophilicity of the solvent.

MATERIALS AND METHODS Materials ct-Chymotrypsin (EC 3.4.21.1., type II from porcine pancreas), N-benzoyl-L-tyrosine methyl ester(Bz-Tyr-OEt), L-arginine (Arg), L-argininamide (Arg-NH2), L-arginine methyl ester (Arg- OMe) and L-arginine ethyl ester (Arg-OEt) were obtained from Sigma Chem. Co., and used without previous purification. Dimethylsulphoxide (DMS), N,N'-dimethyl formamide (DMF), acetonitrile (ACN, acetone (AC) and tetrahydrofurane (THF) were Merck, analytical grade. Celite 545 (0.01 - 0.04 mm particle size) were obtained from Merck. Immobilization process. 40 mg of ct-chymotrypsin were dissolved in 4 ml of 0.1 M phosphate buffer pH 7.8, and mixed with 1 g of Celite. The mixture was shaked during 30 minutes at room temperature and then lyophilized. All the placed protein was adsorbed into the support, measured by the Lowry's method, having the immobilized derivative showed an esterase activity towards N-acetyl-L- tyrosine ethyl ester of 10.45 U/rag support and a water-content of 0.18 mg H20/g support. Kyotorphin synthesis reaction The substrate solution was prepared as follows: 40 I.tmol of Bz-Tyr-OEt and 60 tamol of Arg-OEt were dissolved in 1.9 ml of the assayed water-organic solvent mixture containing 60 ~tmol of triethylamine. Then, into a 2-ml total volume test tube 0.1 ml of water were added to 10 mg of the immobilized derivative, and the reaction was started by the addition of the 1.9 ml of substrates solution. The reaction mixture was incubated with magnetic stirring at 30 oC. Aliquots of 50 lal were extracted at several time intervals from the reaction mixture, and mixed with 100 lal of 10 % (w/v) trichloroacetic acid (TCA) to stop the reaction. TCA-treated samples were diluted with 850 ttl of mobile phase and centrifuged (10 rain. at 2,800 g) at 6 °C to separate the immobilized derivative and then, analyzed by HPLC. IIPLC analysis Substrates and products concentrations were determined by HPLC. A Shimadzu LC-6A chromatograph equipped with a Nova-Pack C-18 column (Millipore, 15 cm length and 3.9 mm internal diameter, 4 lam particle size, and 6 nm pore size), was used. Samples were eluted isocraticaUy with water:acetonitrile:acetic acid (75:20:5. v/v/v) at 1 ml/min, flow rate and the elution profiles detected at 280 nm. One unit of activity was defined as the amount of enzyme which produced 1 ~mol of Bz-Tyr-Arg-OEt product per minute. Measurement of water activity Water activity of the reaction media was determined using an humidity and temperature digital indicator HUMIDAT-IC II (Novasina, Zurich, Switzerland), with a humidity sensor model BS- 3(4)/PP (Novasina). The humidity sensor was checked and periodically recalibrated at three points, with control saturated salt solutions (LiCI, Aw = 0.113; Mg(NO3)2, Aw = 0.544; and BaCI:, Aw = 0.905) for the overall measuring range. Determination of water content The water content of immobilized enzyme preparation in each reaction media was determined as follows: 10 mg of ct-chymotrypsin-Celite complex were suspended into 2 ml of each assayed water-organic solvent mixture, and homogenized during 30 rain. at room temperature. Then, the immobilized derivative was separated by centrifugation, and their water content was measured by the optimized Karl-Fischer method using a moisture titrator MKS-210 (Kyoto Electronics, Japan).

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RESULTS AND DISCUSSION

Influence of nucleophile concentration.

The effect of nucleophile (Nu) concentration on the ct-chymotrypsin activity for the synthesis of

the kyotorphin derivative is shown in Figure 1. Arg, Arg-NH2 Arg-OMe and Arg-OEt were

chosen as nucleophiles in order to analyze the influence of the C%protection group on the

synthetic activity, using a common engineered medium (ACN 90 % v/v) (Reslow et a1.,1988;

Cerovky and Jakubke, 1994). In all cases, the increase in nucleophile concentration was followed

with a higher peptide-synthetic activity to reach a saturation level with a maximum.

m

0 1 2 3 4 [Nu] I [Bz-Tyr-OFt|

Figure 1. Effect of nucleophile (Nu)/Bz-Tyr-OEt concentration ratio on kyotorphin synthesis catalyzed by the immobilized c~-chymotrypsin in acctonitrile:watcr 9" I (v/v) at 30 °C using an initial Bz-

Tyr-OEt concentration of 20 nuM. Nu: Arg (A), Arg-NH2 (1), Arg-OMc (4,) and Arg-OEt (e).

These facts are consequence of the increased rate

of aminolysis with respect to hydrolysis of the

acyl enzyme intermediate, as characterized a

kinetically controlled peptide synthesis process

(Kise et al., 1988, Nagashima et al., 1992, Lozano

et al.,1992). However, the C%protection of the

arginine component with an ethyl ester group

showed the highest synthetic activity, where the

saturation level was reached for a nucleophile

concentration 1.5-times higher than the Bz-Tyr-

OEt. In the Arg-NH2 case, it was found that the

higher nucleophile concentrations inhibit the

reaction rate, as it was also reported for other

C%protection groups (i.e. propyl ester) of the

arginine component (FlOrsheimer et al., 1989).

Effect of water-miscible aprotic solvents

In kinetically-controlled peptide synthesis, the enzyme activity can be enhanced when water

concentration is reduced by the addition of water-miscible organic solvents. Additionally, in all the

solvent-containing biocatalytic system, the nature of the solvent influences the activity and stability of

the enzyme to a large extent (Mozhaev et ai.,1989; Batra and Gupta, 1994; Lozano et al.,1993,

1994). In this order, the influence of five water-miscible aprotic solvents (DMS, DMF, ACN, AC

and THF; log P = -1.35, -1.01, -0.34, -0.24 and 0.49, respectively) on the kyotorphin synthesis

catalyzed by the immobilized ct-chymotrypsin derivative has been studied. As it can be seen in Figure

2, in all cases the addition of organic solvents allowed to increase the synthetic activity of the enzyme

towards a maximum level, which was reduced when the solvent concentrations were increased.

Additionally, the optimum solvent concentration was higher as the solvent hydrophilicity was

605

increased (DMS > DMF >ACN > AC >THF ). Thus, for the most hydrophobic solvent (THF), the

maximum level of activity (2.2 U/g sup.) was found at the lowest assayed solvent concentration (1.2

M), while for the most hydrophilic solvent (DMS), the immobilized derivative showed the highest

absolute activity (10.8 U/g sup.) at a 5.7 M solvent concentration. Obviously, these results should be

explained as a function of both the changes in the macroenvironment of the engineered reaction

media, as well as, the effect of these solvents on the microenvironment of the immobilized

biocatalyst.

The effects of water-miscible aprotic solvents on the engineered reaction media for a peptide-

synthetic reaction catalyzed by an hydrolytic enzyme, such as a-chymotrypsin, are usually attributed

to a mass action effect on water concentration determined by its thermodynamic activity (Aw),

where the synthetic reaction are favoured at lower values by the kinetic control (Blanco et al., 1992;

Lozano et al., 1992, 1993).

20

lOO

so

6o

4o

20

o o 6 10 16

[ SOLVENT ] ( M ) 0.6 0.7 0.8 0.9 1.0

Figure 2. Effect of solvent concentration on Bz- kyotorphin synthesis catalyzed by the immobilized c~- chymotrypsin derivative, using Bz-L-TyrOEt (20 mM) and L-ArgOEt (30 raM) as substrates at 30 °C. DMS (1), DMF (O), ACN (O), AC (O) and THF (A).

12

1: & 9 Q.

14

.? = 6 >. i -

_5 )-- 3 o <

0 0.5

v

F-

Aw

Figure 3. Dependence of the Bz-kyotorphin synthesis ~mlyz~ by the immobilized ct-chymotrypsin derivative with the water activity of the reaction media, using Bz-L- TyrOEt (20 raM) and L-ArgOEt (30 raM) as substrates at 30 °C, and different organic solvents DMS (ll), DMF (Q), ACN (O), AC (I-I) and THF (A).

In this order, Figure 3 shows the synthetic activity of the immobilized ct-chymotrypsin derivative as a

function of the Aw in the reaction media induced by the presence of these aprotic solvents at several

concentrations. In all cases, firstly, a clear enhancement of the peptide synthesis activity was

observed when the Aw was reduced from 1.0 (100 % aqueous media) to an optimum value for each

606

assayed solvent. This fact was explained by the increase in the water-solvent interactions, shifting the

equilibrium towards synthesis. ARerly, as the Aw parameter decreases, the activity of the

immobilized biocatalyst become less, probably due to direct effects of solvents on the active enzyme

conformation or to the disruption of water structure in the vicinity of the active site (Mozhaev et

al., 1988, Batra and Gupta, 1994).

On the other hand, the increase in solvent hydrophilicity (or decrease in log P) allowed to reduce the

optimum Aw of the reaction media, as well as, to increase the maximum level o f activity of the

immobilized derivative. These results could be attributed to the microenvironmentai effects of these

solvents on the enzyme-water interactions, and could be studied as a function of the changes in

water-content of the immobilized derivative (Reslow et al., 1988) (see Figure 4). At it can be seen, in

all cases, the decrease in water-content of the immobilized derivative from 2.18 nag water/g support

(corresponding to a 100 % aqueous media) enhanced the synthetic activity to an optimum value for

each assayed solvent. From an overall point of view, the more solvent hydrophilicity increases (or

log P decreases), the more optimum water-content of the immobilized derivative is reduced, and the

more synthetic activity is enhanced. Additionally, these results also show that the increase in solvent

hydrophobicity enhanced the denaturing efficiency of the organic solvent, probably due to its binding

proceeds with hydrophobic residues of protein.

Furthermore, it is remarkable that the synthetic activity of the immobilized derivative showed a

12

i $

g (3 3 <

0 0.0 0.6 1.0

t 1 1.6 2.0

WATER CONTENT (rng HzO/g support)

Figure 4. Effect of water content of the immobilized ct- chymotrypsin derivative on Bz-kyotorphin synthesis, using Bz-L-TyrOEt (20 raM) and L-ArgOEt (30 mM) as ~ - a t e s at 30 °C. DMS (1), DMF (e) , ACN (O) and

THF (A).

1.0 2.0

jo / 1'"! 0.6 1.2 o

-2 -1 0 1 Log P

Figure 5. Relationship between both the optimum Aw (e ) of the reaction media and the water content of the immobilized derivative (11) with the hydrophilicity of the solvent measured by the log P parameter

607

similar behaviour respect to both the macro- and microenvironmental water parameters, where the

optima conditions for Aw and water-content were stablished by the highest synthetic activity. These

facts are clearly show in Figure 5, where these optima water parameters obtained from Figures 3 and

4, respectively, are depicted as a function of the log P of the solvent. In this Figure, both water

parameters of the system shows a saturation profile, where the reduction in solvent hydrophilicity

increases the optimum Aw of the media and the water-content of the immobilized derivative.

Reslow et al., (1988) reported the importance of the support material for bioorganic synthesis as a

function of the influence of water partition between solvent, enzyme and solid support. These

authors concluded that the low aquaphilicity of Celite (amount of water on the support / amount of

water on the solvent) allowed to obtain the best results in an amino acid ester synthesis catalyzed by

ct-chymotrypsin in organic media at low water content (<3 %). In our case, this low aquaphilicity

should be observed as a weak protective effect of the support towards the direct enzyme-solvent

interactions, being reduced the denaturative effect of solvent when its hydrophilicity (adequately

measured by the log P parameter) was increased.

In conclusion, this works clearly shows the fact that solvent hydrophilicity is an extremely important

when optimizing biocatalytic system for peptide synthesis in organic media, being the Aw of the

media and the water content of the biocatalyst the most important parameters. The increase in

solvent hydrophilicity allows a possitive modification of both the macro- and microenvironment of

the immobilized c~-chymotrypsin to carry out synthetic reactions.

ACKNOWLEDGEMETS.

This work was partially supported by the "Consejeria de Cultura, Educacion y Turismo. Comunidad Autrnoma de la Region de Murcia" grant n ° PTC 93/27. Spain.

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