Enzyme reaction engineering: Design of peptide synthesis by stabilized trypsin

Enzyme reaction engineering: design of peptide synthesis by stabilized trypsin Rosa M. Blanco, Gregorio Alvaro and Jos~ M. Guisan lnstituto de Cattilisis, C.S.I.C., Madrid, Spain

By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS)). In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of l80 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst.

Keywords:Enzymesin organic synthesis;designof kineticallycontrolledsynthesis;peptide synthesisby stabilized trypsin; design of thermodynamicallycontrolled synthesis;enzymesin organic solvents

Introduction

Address reprint requests to Dr. Guis~.nat the lnstituto de Cat~tlisis y Petroleoquimica,C.S.I.C., Serrano 119, 28006 Madrid, Spain Received 26 April 1990; revised 21 November 1990

parameters may be quite different and even opposite. Hence, optimal industrial conditions should be established as a compromise solution which includes a set of variables corresponding to good values, but likely nonoptimal ones, for all parameters of interest. In addition, some enzymatic reactions of industrial interest, such as peptide synthesis by proteases, can be performed by following different synthetic strategies (kinetically controlled synthesis in aqueous media, equilibrium controlled synthesis in water-organic cosolvent systems, equilibrium-controlled synthesis in biphasic systems). Therefore, compromise solutions corresponding to each strategy should be compared in a very integrated way in order to obtain the best conditions to carry out a synthetic process at industrial scale. Thus a very strong relationship between the preparation of enzyme derivatives and the enzyme engineering reaction can be established. If we are able to prepare very active and very stable enzyme derivatives, we shall be able to use simpler synthetic strategies and more drastic experimental conditions. Evidently, the optimal conditions for a given reaction (best synthetic strategy and best experimental conditions) will be very

© 1991 B u t t e r w o r t h - H e i n e m a n n

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Proteases present extraordinary oportunities to be used as catalysts of peptide synthesis; because of their impressive specificity (towards substrate, stereospecificity, regiospecificity), the protection of lateral chains is usually unnecessary and risk of racemization of amino acids is minimal. However, enzymes are very labile catalysts, and so the design of industrial enzymatic reactions--enzyme reaction engineering--must be done very carefully. When we design a practical enzyme reaction, we have to study the effect of the different variables which define the reaction medium (pH, temperature, presence of organic solvents) on every parameter of industrial interest (activity/stability of the catalyst, solubility of reactants, stability of reactants and products, yields, etc). Obviously, the effect of each variable on different

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Papers related to the properties of the particular derivative used as catalyst. By using a strategy of immobilization/stabilization of enzymes through multipoint covalent attachment to activated preexisting supports,l we have been able to prepare very active and very stable trypsin-agarose derivatives: 50 mg of pure enzyme per milliliter of packed catalyst with 100% immobilization yield (immobilized enzyme/offered enzyme), retention of 70% of catalytic activity (corresponding to soluble enzyme that has been immobilized), and thermal stability increased 12,000-fold with relation to soluble in the absence of autolysis phenomena or in relation to onepoint attached derivatives.~-3 In this paper we present studies of reaction engineering of the synthesis of the model peptide benzoyl arginine leucinamide using these stabilized trypsin derivatives as catalysts. We have tested two different synthetic strategies: (i) kinetically controlled synthesis in aqueous media by using benzoyl arginine ethyl ester and leucinamide as substrates and (ii) equilibrium controlled synthesis in water-organic cosolvent monophasic systems by using benzoyl arginine and leucinamide as substrates. We have mainly tested some variables (e.g. polarity of the reaction medium, exact structure of the enzyme derivative) which are usually not well studied in scientific reports related to this synthetic field. Optimal values of other more common variables (pH, nature and concentration of nucleophile, nature of the organic cosolvents) were established in previous studies performed in our laboratory, which will be reported in forthcoming papers. 4 in the present paper, we shall comment on the optimization of each synthetic strategy, and finally we shall present an integrated comparison between these two strategies. Both synthetic approaches were carried out in monophase systems, because the use of biphasic ones (with immiscible organic solvents) seemed to be difficult and not promising in this case, as a consequence of the great hydrophilicity of trypsin substrates.

Kinetically controlled synthesis Although the fundamentals of this synthetic approach have been extraordinarily well described and discussed by Kasche, 5 we shall briefly describe this approach mainly from the point of view of enzyme reaction engineering. In a very simplifed way, the scheme of this synthetic approach can be represented as follows:

There is a crucial step in these kinetically controlled syntheses in which the current ratio aminolysis/hydrolysisj is established. This step occurs when the ester, benzoyl arginine ethyl ester (BAEE), reacts with the enzyme and a covalent acylenzyme complex is formed. Then, if the enzyme molecule has a nucleophile molecule adsorbed on another close area, subsite Sj', 6 of its active center, a competence between dissolved water and the adsorbed nucleophile molecule is established in order to nucleophilically attack the acylenzyme complex. Hence, this reaction yields peptide (synthesis or aminolysis), when the attack is performed by the nucleophile amino acid amide, or benzoyl arginine (BA) (hydrolysis 0 when water acts as the nucleophilic agent. On the contrary, if there are no nucleophile molecules adsorbed on the active center of the enzyme, only the hydrolysis~ can occur. From a practical point of view, it would be very interesting to get the highest aminolysis/ hydrolysis ratio and hence the highest synthetic yields by using the least possible excess of nucleophile. Therefore, it is important to establish the experimental conditions in which this adsorption equilibrium of the nucleophile on the active center of the concrete enzyme derivative is favored. In addition to this critical event, there are a number of parameters which are also very important for the design of the industrial performance of this synthetic reaction: Time course of the synthetic reaction, which can be represented by the initial synthetic rate plus the linearity of the whole time course. To define linearity, we can use the parameter r as defined by Carleysmith et al.7: r = the ratio between the actual time necessary to reach the highest synthetic yield and the time necessary if the reaction rate were constant and equal to the initial one. Highest synthetic yield reached during a whole timecourse of kinetically controlled synthesis, hereinafter synthetic yield. This is defined by a complex combination of initial aminolysis/hydrolysis ratio, possible variation of this ratio due to several factors (conformational changes of the enzyme during the whole time course, depletion of nucleophile), and rate of peptide hydrolysis (hydrolysis:). Kinetic stability of the synthetic product (which is thermodynamically instable). This can be defined by the ratio between the rate of peptide synthesis and the rate of peptide hydrolysis (hydrolysisz). Stability of the enzymatic derivative.

Synthesis (aminolysis)

Benzoyl arginine ethyl ester + LeuNH 2 + H20

Hydrolysis1

~

BALeuNH 2 + EtOH + H20

Trypsin

Benzoyl arginine + EtOH + LeuNH 2

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SiS 2

Peptide synthesis by stabilized trypsin: R. M. Blanco et al. Thermodynamically controlled synthesis From different points of view--theoretical, practical, and economic--"thermodynamically controlled synthesis" in monophasic water-cosolvent systems is a priori the easiest strategy to synthesize peptide bonds by proteases: (i) This is a direct reaction between an acid and an amine group to yield an amide bond. (ii) The product of the reaction is fully stable (both kinetically and thermodynamically). (iii) The activation of the acyl donor becomes unnecessary. (iv) The pH values and water activities are easily controllable. (v) The synthetic yield corresponding to the most expensive component can be increased to nearly 100% by using an excess of the most inexpensive, most soluble, most easily separable reactant. (vi) The subsequent purification steps may be quite simple: we only need to separate the synthetic product from the component in excess. Although this equilibrium controlled strategy has been poorly developed, there are a few very interesting related papers with the fundamentals of this strategy. 5'8-~°. We shall briefly discuss this strategy, having in mind the practical aspects of the reaction design. In a simple way, the scheme of this synthetic approach can be represented as follows: Trypsin

R-COOH

R - COO

+ NH z - R '

~

~ R-CONH-R'

+ H20

The use of organic cosolvents greatly increases these synthetic yields. The main effects of the presence of organic cosolvents are (i) Reduction of aw, which is more intense when the concentration of the cosolvent increases, and (ii) stabilization of the non-ionic forms of the ionizable groups, mainly the acid group (e.g. the pK of acetylglycine in 80% DMSO is increased to 6.931°)). Thus, from a thermodynamic point of view, the use of high concentrations of very apolar cosolvents is absolutely essential to produce a dramatic improvement in the performance of this synthetic reaction. However, this is the main drawback of this synthetic approach: The presence of high concentrations of apo-

lar cosolvents may exert very important deleterious effects on the activity and stability of the enzyme derivative. These effects may be quite complex: (i) modification of the three-dimensional structure of the enzyme derivative and (ii) direct interaction of the organic cosolvent with the active center of the enzyme, promoting changes in pKs of ionizable groups essential for the catalytic action of the enzyme and/or adsorbing on recognition sites and competing with adsorption of substrates on the active center of the enzyme. Because of these complex and opposite effects, it is evident that, from a practical point of view, equilibrium controlled synthesis in water-cosolvent systems must be very carefully designed. As remarked above, it is necessary to perform an integrated study of the effect of the composition of the reaction medium on very different parameters of industrial interest. The main parameters to be tested are:

+ NH~ - R '

Since only the non-ionic forms of both acid and amine are involved in the synthetic reaction, 5 the equilibrium constant, Kth, may be represented as: [R - CONH - R']

K,h/aw = [R - COOHI[NHz - R']

[1]

From this equation and according to ref. 8, the peptide concentration in the equilibrium may also be represented as a function of the total concentrations of acid and amine: [peptide] = KthK,o,_ion[acid][aminel/aw

Time course of the synthetic reaction (enzyme activity, concentration of reagents, inhibition byproducts) Enzyme stability Thermodynamic synthetic yield Solubility and stability of substrates and product

[21

where Knon.io n iS the ratio between the product of the concentrations of the non-ionic forms and the product of the total concentrations of acid and amine. Hence this constant will be related to the pH and the pKs of the amine and acid groups. In a fully aqueous medium, aw is very high and Knonion at all pH values is very low. The latter is a consequence of the great difference between the pKs of the amine and carboxy terminal groups of amino acids (e.g. the pKs corresponding to the carboxy and amine groups of glycine are 2.35 and 9.7811). The value of ApK is greatly reduced when using protected amino acids (e.g. ~pK between acetylglycine and glycinamide is 4.61° instead of 7.43 for unprotected glycine), but it continues to be very high. As a result, synthetic yields in fully aqueous media are negligible.

Materials and methods

Materials Sepharose CL 6B gels were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. The enzyme trypsin (E.C. 3.4.21.4) from bovine pancreas, BAEE (benzoyl arginine ethyl ester), BA (benzoyl arginine), and LeuNHz (leucinamide) were purchased from Sigma Chemical Co., St. Louis, MO. Trypsin was a Type III Sigma preparation, dialyzed and lyophilized. The specific hydrolytic activity of this preparation is 10,000-14,000 BAEE units per milligram of protein.

Preparation o f trypsin (amine)-agarose (aldehyde) derivatives One-point and two different multipoint covalent attached trypsin-agarose derivatives were prepared as previously described, j The main characteristics of these derivatives are given in Table 1. In general, we have used very high-loaded trypsin derivatives containing 50 mg of pure enzyme per milliliter of catalyst. However, in some cases, e.g. the very fast kinetically

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Papers Table 1 Main characteristics of trypsin-agarose derivatives used in the present study Derivative a

Enzyme loading b

Activity c

Stabilization d

A1 E3 E5

20 50 50

100 100 70

1 5,000 12,000

a Experimental procedure for preparation of these derivatives is briefly described in the text and detailed in refs. 1-3 b Enzyme loading is expressed as milligrams of pure enzyme per milliliter of derivative. A1 presents a lower enzyme loading because of reversibility of one-point enzyme support attachment 1 c Activity is expressed as percentage corresponding to soluble enzyme that has been immobilized d Stabilization is the ratio between half-life of each derivative and the corresponding one to soluble enzyme in the absence of autolysis phenomena 2

controlled synthesis (KCS), we have also used very low-loaded trypsin derivatives (0.5 mg of pure e n z y m e per milliliter of packed catalyst) in order to prevent diffusional limitations which may influence the apparent behavior of the immobilized enzyme.

Synthetic reaction

Kinetically controlled synthesis. This reaction was performed in a small beaker very gently stirred inside a high-low temperature incubator. The assay mixture was constituted by l0 ml of 0.1 M borate buffer pH 9.0 with 20 mM of both reactants, B A E E and LeuNH~. The reaction was started by adding 350 mg of wet derivative (corresponding to 0.5 ml of packed gel and hence to 0.25 mg of immobilized trypsin). Different variables were tested: (i) temperature between 4°C and 37°C, (ii) ionic strength between 0.1 M borate and 3 M ammonium sulfate, and (iii) presence of organic cosolvents between fully aqueous medium to 85% organic cosolvent. Thermodynamically controlled synthesis. The reactor was a thermostated jacket column packed with 10 ml of our trypsin-agarose derivatives. Before starting the reaction, the column was equilibrated by fluxing 50 ml of the water-organic cosolvent mixture. The reaction mixture was as follows: 100 mi 7 m M B A and 20 mM LeuNH~ dissolved in different w a t e r - c o s o l v e n t mixtures at pH 7.0 (no buffer was used because the nucleophile acts as a buffer in the range of pH values studied). For kinetic analysis, the reaction mixture was pumped through the column at different flow rates (from 0. I to 2 ml min -I) and aliquots of the eluted solution were analyzed by H P L C . Reaction rates were obtained from plots of percent conversion versus residence times. For equilibrium analysis, the reaction mixture was fluxed through the column at a constant flow rate (0.7 ml min 1) and the first 50 ml eluted out from the column was discarded. Then the reaction mixture was recirculated through the column at the same flow rate with continuous external adjustment of pH by using 1 M

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N a O H as titrant. Corresponding amounts of cosolvent were also added (e.g. a volume of cosolvent per volume of titrant when the cosolvent concentration was 50%) in order to keep the cosolvent concentrations constant. The use of highly concentrated titrant solution avoided excessive dilution of the reaction mixture (the volume was increased by less than 5%) and so dilution of reactants was minimal.

HPLC analysis Substrates (benzoyl arginine or benzoyl arginine ethyl ester) and products (peptide and also benzoyl arginine in kinetically controlled synthesis) were separated and quantified by reverse phase H P L C . At different times, aliquots of the supernatants [in KCS or in thermodynamically controlled synthesis (TCS)] were withdrawn and diluted with 4 volumes of mobile phase, filtered through a Millipore filter of 0.45 /zm, and analyzed by reverse phase H P L C . A Konik Instruments (San Cugat, Spain) with a Spectra Physics SP 8450 UV detector and a 250 x 4.6 mm RP-C 18 (5 /~m) column (Spherisorb) were used. Samples were eluted isocratically with 40% ethanol, 60% water containing 0.1% phosphoric acid as the mobile phase in kinetically controlled experiments, and 50% ethanol, 50% water containing 0.1% phosphoric acid in thermodynamically controlled synthesis. The flow rate was 1 ml min i and the amount of reactant and products was determinated from calibration curves using stock solutions.

Stability o f Trypsin-agarose in 1 M ammonium sulfate Samples of Al and E5 derivatives (see Table 1) were suspended in 0.1 M borate buffer containing 1 M ammonium sulfate and incubated at 4°C and 25°C. At different times, aliquots of these suspensions were withdrawn and assayed by following the hydrolysis of B A E E in the absence or presence of I M ammonium sulfate using a simple spectrophotometric assay previously described. 1 In certain cases, parallelism between decay of hydrolytic and synthetic activity (KCS) was also checked by using the synthetic assay described above.

Stability o f trypsin-agarose in the presence o f cosolvent Samples of A1 and E5 derivatives (see Table l) were suspended in 20 mM leucinamide (as buffer) in 90% cosolvent mixture at 25°C. At different times, aliquots of this suspension were withdrawn and assayed for B A E E hydrolysis as mentioned above. Two different assays were performed in each case: (i) An assay was performed at pH 7.5, 25°C in fully aqueous medium. This tests for the irreversible loss of catalytic activity. (ii) An assay was also performed at pH 7.5, 25°C in 90% cosolvent, which tests the total loss of catalytic activity that occurs when the e n z y m e is acting in optimal conditions of equilibrium controlled synthesis. In certain cases, parallelism between the decay of by-

Peptide synthesis by stabilized trypsin: R. M. Blanco et al. drolytic and synthetic activity (TCS) was also checked by incubating great amounts of derivative and performing the synthetic kinetic assay described above.

Results and discussion

Kinetically controlled synthesis Effect of temperature. Synthetic yields greatly increase at low temperatures (Figure la). In Figure lb we represent a graphic example of the whole time courses of peptide synthesis obtained at two very different temperatures, 4°C and 37°C. In addition, quantitative data of the different reaction rates obtained at 4°C and 37°C are given in Table 2. Rates of hydrolysis of the acyl enzyme complex first (hydrolysis 1or initial BA production), and secondly ofpeptide hydrolysis (hydrolysise), increase with temperature much more than the rate of aminolysis of the acyl enzyme complex (peptide synthesis). Hydrolytic rates increase by a factor of 8.3 and 12.5 from 4°C to 37°C (Table2), which agrees very well with average activation energies found for enzymatic catalysis (a twofold increase in catalytic rates per 10oc).12 However, the increase of synthetic rate is too low, only a 2.9-fold factor. Since we are using nonsaturating nucleophile concentrations, we might assume that the very low increase in synthetic activity may be mainly related to a decrease of the adsorption constant of the nucleophile to the enzyme when temperature increases. At high temperatures, if the concentration

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Figure 1 Effect of temperature on kinetically controlled synthesis of BALeuNH 2 catalysed by stabilized trypsin-agarose. (a) Maximal synthetic yields (synthetic yields) obtained at different temperatures. (b) Time course of peptide formation and hydrolysis at 4°C (A) and 37°C (O). Experiments were performed in standard conditions described in Methods at pH 9.0 and in the absence of cosolvents or ammonium sulfate. Synthetic yields are expressed as percentage of activated acyl donor (BAEE) transformed in peptide.

Table 2 Kinetic parameters corresponding to synthesis performed at 4°C and 37°C Temperature

Synthesis

Hydrolysis 1

4 37 ratio 37/4

0.2 0.58 2.9

0.23 1.9 8.3

Vs/Vhl Hydrolysis2 0.9 0.3 0.33

0.008 0.1 12.5

Rates are expressed as mmol min -~ in the standard conditions described in Methods. Experiments were performed in the absence of ammonium sulfate at pH 9.0

of trypsin molecules containing leucinamide adsorbed on the active center is much lower than at low temperatures, this will result in synthetic rates that do not increase in proportion to the increase on temperature. Adsorption of leucinamide on the active center of trypsin is a hydrophobic process. 13It has been reported that hydrophobic interactions are favored when temperature increases. 14However, we should assume that hydrophobic interaction is perhaps the least important factor to consider when discussing hydrophobic adsorption. The major role of thermodynamics in these adoptions corresponds to the changes in solvation energies of both the hydrophobic subsite of trypsin surrounded by water and the hydrophobic moiety ofleucinamide dissolved in water. Since solvation of hydrophobic compounds is favored when temperature increases, we might initially assume that the overall thermodynamics of this hydrophobic adsorption is exothermal, exactly the opposite than the concrete hydrophobic interaction. From a practical point of view, whatever the correct scientific explanation of the experimental results is, it seems very clear that by lowering temperature, we greatly improve the performance of this synthetic reaction as it has been graphically represented in Figure lb. The synthetic rate decreases only by a factor of 2.9, but the synthetic yield increases considerably (from 20% to 40%). In addition to that, the kinetic stability of the synthetic product (in terms of rate of synthesis/ rate of hydrolysis2) greatly improves (approximately a fourfold factor). On the other hand, the linearity of the whole time course of synthesis is excellent; r value is less than 2 (r = t/q in Figure lb). This small decrease in synthetic rate (Figure lb) as well as the aminolysis/ hydrolysis ratio (results not shown) seems to be due only to the significant depletion of the nonsaturating nucleophile concentration as a consequence of the synthetic process (we were using equimolar concentrations of both acyl donor and nucleophile). These stabilized trypsin-agarose derivatives seem to behave as quite rigid catalysts during the whole time course of this synthetic process.

Effect of polarity of the reaction medium. (1) Presence of ammonium sulfate. The presence of high concentrations of this salt in the reaction medium promotes a

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3O

2O

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Figure 2 Effect of the presence of high concentrations of a m m o nium sulfate on m a x i m a l synthetic yields obtained during the time course of kinetically controlled synthesis of BALeuNH2 catalysed by stabilized t r y p s i n - a g a r o s e . Experiments were carried out in standard conditions described in Methods at 4°C and pH 9.0. Synthetic yields are expressed as percentage of activated acyl d o n o r (BAEE) transformed in peptide

significant increase in synthetic yields (Figure 2). The highest value is reached at 1 M concentration. As the concentration continues to increase, the synthetic yields decrease. They do, however, always remain higher than the yields obtained in the absence of this salt. In Table 3 we compare the different parameters involved in this kinetically controlled synthesis obtained in the absence of ammonium sulfate and in the presence of the optimal concentration, 1 M. We observe that the rate of hydrolysis of the acyl enzyme complex remains practically constant but the rate of peptide synthesis greatly increases. Since ammonium sulfate exerts a very important "salting out" effect, it seems to be logical that the presence of ammonium sulfate may improve the thermodynamics of this hydrophobic adsorption between the nucleophile and the active center of trypsin. So in the presence of this salt a higher concentration of trypsin molecules has adsorbed the nucleophile at the active center, in these nonsaturating nucleophile concentrations. In addition to that, the presence of ammonium sulfate also promotes an additional decrease in the rate of peptide hydrolysis. This results from an increased kinetic stability of the synthetic product (rate of synthesis/rate of hydrolysis2 increases by 3.5-fold). This fact and the decrease of synthetic yields with even higher ammonium sulfate concentrations could be explained through small conformational changes in the exact structure of the active center of the enzyme promoted by the presence of these high salt concentrations. From a practical point of view, the use of this high concentration of ammonium sulfate results in higher yields: synthetic rate and synthetic yield increase and, on the other hand, the rate of hydrolysis of the synthetic product suffers an additional significant decrease (the resulting peptide is kinetically more stable). The linearity of the time courses of the reaction continues to be excellent (results not

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shown) and, as we shall comment later on, the stability of our E5 derivative is also extraordinary. (2) Presence of organic cosolvents. We have tested the effect of the presence of very high concentrations of organic cosolvents (polyethyleneglycol, 1 : ! polyethyleneglycol-ethanol, and butanediol), on the whole time course of this synthetic reaction. Both the rate of initial hydrolysis of ester and the rate of hydrolysis of the peptide decrease dramatically in the presence of organic cosolvents (Table 4). However, the rate of peptide synthesis decreases even more significantly. This effect seems to be more acute when the polarity of the cosolvent decreases. In addition to possible conformational changes induced by the presence of this high concentration of organic cosolvent, we may also assume that the presence of the cosolvent must affect the thermodynamics of hydrophobic adsorption of leucinamide on the trypsin active center. The cosolvents should promote a decrease of the concentration of trypsin molecules having nucleophile hydrophobically adsorbed on their active centers. This would result in a decrease in the ratio synthesis/hydrolysis. Effect of the current structure of the catalyst. We have

also tested the behavior of other two trypsin-agarose derivatives, A1 and E3 (see Table 1), as catalysts of this kinetically controlled synthesis in two different experimental conditions: in standard conditions used by other authors to carry out similar syntheses, e.g. at

Table 3 Kinetic parameters corresponding to synthesis perf o r m e d in aqueous m e d i u m or in the presence of 1 M a m m o n i u m sulfate [(NH4)2SO 4]

Synthesis

Hydrolysis1

0 1 ratio 1/0

0.2 0.34 1.7

0.23 0.27 1.15

Vs/Vhl Hydrolysis2 0.9 1.37 1.5

0.008 0.004 0.5

Rates are expressed a s m m o l min l in the standard conditions described in Methods. Experiments were performed at 4°C and pH 9.0

Table 4 Kinetic parameters corresponding to kinetically controlled synthesis performed in the presence of high concentrations (85%) of different organic cosolvents Cosolvent -1 : 1 PEG 600 + ethanol PEG 600 Butanediol

Vs/Vhl Hydrolysis2

Synthesis

Hydrolysis 1

0.8 0.16

1.3 0.05

0.5 0.32

0.05 --

0.023 0.01

0.09 0.05

0.25 0.2

---

Rates are expressed as m m o l min -1 in the standard conditions described in Methods. Experiments were performed at 25°C and pH 9.0. Rates of hydrolysis of peptide (hydrolysis 2) were negligible in the presence of organic cosolvents

Peptide synthesis by stabilized trypsin: R. M. Blanco et al. Table 5 Effect of the current structure of trypsin derivatives on their catalytic behavior in kinetically controlled synthesis

Derivativea

Synthetic yield in standard conditions b

Synthetic yield in optimal conditions c

A1 E3 E5

37 34 27

45 49 54

a Characteristics of each derivative are given in Table 1 b Synthetic reaction was performed at 25°C in 0.1 M borate buffer pH 9.0 c Synthetic reaction was performed at 4°C in 0.1 M borate and 1 M a m m o n i u m sulfate pH 9.0

tive was much more stable than the A l derivative in the presence of this high salt concentration. The E5 derivative remains fully active after 60 days' incubation in the optimal conditions of this synthetic reaction. Even irreversible inactivation is practically negligible after 60 days' incubation at 25°C, which suggests that at the optimal 4°C, full activity will be preserved after much longer than 60 days. This derivative obviously presents very important prospects in industrial scale reactions for this type of synthetic approach. In comparison, the one-point attached derivative loses a high percentage of activity after incubation at both 4°C and 25°C (Figure 3).

Thermodynamically controlled synthesis 25°C in 0. I M borate buffer pH 9.0, and in the optimal conditions found in this work for E5 derivative, 4°C and presence of 1 ~l ammonium sulfate in the reaction mixture at pH 9.0 (Table 5). E5 derivative performs badly as a synthetic catalyst in standard conditions: the synthetic yield is 27%, and that corresponding to the A1 derivative (with structure presumably equal to soluble enzyme) is 37%. However, under optimal conditions, our very stable E5 derivative is the best synthetic catalyst. Synthetic yields, using this nonsaturating nucleophile concentration, increase to 54%. The less stabilized E3 derivative behaves in an intermediate way. It seems that the very intense enzyme support multipoint attachment which yields the E5 derivative 2 promotes slight conformational changes in the enzyme structure which result in the loss of hydrolytic activity in E5 (Table 1), as well as the deleterious change in its synthetic behavior in standard conditions. However, the resulting extraordinary rigidity of its three-dimensional structure protects its active center from the important deleterious effects of ammonium sulfate. Hence, the presence of this salt, which improves the hydrophobic adsorption of the nucleophile on the active center of trypsin, has an overall positive effect on the synthetic behavior of the derivative E5. On the other hand, A1 and E3 derivatives preserve full hydrolytic activity (Table 1) and result in better synthetic catalysts in standard conditions. However, they do suffer deleterious effects from ammonium sulfate which mask the beneficial effect of high ionic strength on nucleophile adsorption. Hence these less rigid derivatives, mainly the A1 derivative, result in worse synthetic catalysts in optimal, more drastic, experimental conditions.

Effect of concentration of eosolvent. When the concentration of cosolvent was increased, synthetic yields dramatically increased (Figure 4), as predicted from a thermodynamic point of view. In parallel, the synthetic activity of our E5 derivatives greatly decreases, as we predicted above. In this case, the use of 90% organic cosolvent seems to be a good compromise solution for an adequate design of this reaction. At this cosolvent concentration, synthetic yields were very high (86%)--only a slight increase is further observed when cosolvent concentration is 95%--and synthetic rates are low. They do preserve a significant value, !0 p.mol peptide per hour per milligram of immobilized trypsin, however. In addition to that, the linearity of the whole time course of synthesis in these experimental conditions is very good. The ~- value (defined above) is now less than 2. Initially, the parallel effect of the increase of

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Stability of trypsin-agarose in 1 M ammonium sulfate. We have tested the stability of A1 and E5 derivatives in 0.1 M borate buffer, 1 M ammonium sulfate, and at both 4°C and 25°C. Total and irreversible inactivations were measured. Total inactivation was evaluated by performing tests on residual activity in the presence of 1 M ammonium sulfate, and irreversible inactivation was evaluated by testing residual activity in fully aqueous medium. In Figure 3 we observe that our E5 deriva-

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Time, days Figure 3 Time courses of total and irreversible inactivation of one-point and very intensively multipoint covalent attached trypsin-agarose in the presence of 1 M a m m o n i u m sulfate. Experiments were carried out in standard conditions described in Methods. Squares: E5 derivative. Circles: A1 derivatives. Closed symbols: total inactivation at 25°C. Open symbols: irreversible inactivation at 25°C. Half-filled symbols: total inactivation at 4°C

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Papers cosolvent concentration on stability of the E5 derivative was not checked, since we had results confirming the extreme stability of this derivative, even in more drastic experimental conditions, e.g. 90% dioxane, j5 Effect o f t e m p e r a t u r e . Figure 5 shows the effect of temperature on the yields and kinetics of this reaction. Yields are really thermodynamic equilibrium ones: (i) they were independent of the derivative used, and (ii) they were dependent on substrate concentration according to thermodynamic predictions. 4 These equilibrium yields rise as temperature decreases, indicating that this synthetic process is an exothermic one. This is consistent with data reported in the literature 5'~6'~7 and with other data obtained in our laboratory for similar condensation processes (e.g. cephalothin synthesis catalysed by penicillin G acylase), m The synthetic rates increase with temperature up to 25°C, as expected, with free e n z y m e not suffering any structural modification. H o w e v e r , the reaction rate drops from 25°C to 37°C; this may in fact be due to conformationai changes on the e n z y m e structure at the higher temperature and higher concentrations of organic cosolvent. Again, a compromise "practical solution" is necessary. From data represented in Figure 5, 25°C seems to be the most favorable temperature. Synthetic yields

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I

20

I

30

i

40

TEMPERATURE, °C

Figure 5 Effect of temperature on synthetic rate and on synthetic yields of equilibrium-controlled synthesis of BALeuNH 2 catalysed by stabilized trypsin-agarose. Experiments were performed as described in Methods at pH 7.0 in 90% cosolvent-water mixtures and by using 7 mM of benzoylarginine and 20 mM of leucinamide. Yields (©) are expressed as percentage of acyl donor transformed into peptide. Synthetic rates (0) are expressed as micromoles of peptide per hour per milligram of immobilized trypsin

-- 1000

IOO:

are reduced only slightly over those obtained at lower temperatures, but synthetic rates are quite superior. 100

uJ >-

5O

t

20

I

~

40

I

I

60

t

I

80

J

100

COSOLVENT, %

Figure 4 Effect of concentration of organic cosolvent (a 2 : 1 mixture dioxane-butanediol) on synthetic rate and on synthetic yields of equilibrium-controlled synthesis of BALeuNH 2 catalysed by stabilized trypsin-agarose. Experiments were performed as described in Methods at pH 7.0 and 25°C and by using 7 mM of benzoylarginine and 20 mM of leucinamide. Yields (0) are expressed as percentage of acyl donor transformed in peptide. Synthetic rates, V0, are expressed as micromoles of peptide per hour per milligram of immobilized trypsin. (111)Synthetic rates corresponding to one-point attached A1 derivative; (©) rates corresponding to multipoint attached E5 derivative

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E n z y m e M i c r o b . T e c h n o l . , 1991, v o l . 13, J u l y

Activity/stability o f different t r y p s i n - a g a r o s e derivatives. In Figure 4 we can also compare the effect of concentration of organic cosolvent on the activities corresponding to our very stabilized E5 trypsin derivative and to the one-point attached A1 derivative. At very low cosolvent concentrations, A1 is slightly more active than E3, as we have also observed in hydrolytic reactions (see Table 1). H o w e v e r , when concentrations of cosolvent increase, both derivatives lose a very important percentage of activity. The more rigid E5 derivative remains much more active than unmodified trypsin. Therefore, in the standard conditions to perform this synthetic reaction (7 mM BA, 20 mM LeuNH2, 90% cosolvent, 25°C, pH 7.0), the initial reaction rate obtained with our very stabilized trypsin agarose derivative was 9.0 ~mol peptide per hour per milligram of immobilized trypsin. The initial reaction rate obtained with one-point attached trypsin-agarose was fivefold lower, i.e. 1.8 ~mol peptide per hour per milligram of immobilized trypsin. We have also tested the stability of these two very different trypsin derivatives in these optimal conditions for the synthetic reaction (Figure 6). The multipoint covalent attached derivative is much more stable than the one-point attached trypsin, both for total and for irreversible inactivation in the presence of organic co-

Pepttde synthesis by stabilized trypsin: R. M. Blanco et al. 100

50

g

I

I

20

I

I

I

40

I_

60

T I H E , days

Figure 6 Time courses of total and irreversible inactivation of one-point and m u l t i p o i n t covalent attached t r y p s i n - a g a r o s e derivatives incubated in 90% of c o s o l v e n t - w a t e r mixtures. Experiments were performed as described in Methods. (©) Multipoint attached E5 derivative; (O) one-point attached A1 derivative

solvents. From a practical point of view, both time courses of inactivation are very important. Total inactivation defines the real inactivation course of the derivative when continuously incubated in the reaction conditions. However, if we washed the derivatives with fully aqueous medium, for example after a given number of reaction cycles, we would be able to recover the activity represented in irreversible inactivation time courses. Thus, irreversible inactivation reflects the real industrial possibilities of enzyme derivatives (periodical washings should not be a limiting factor), and total inactivation reflects the complexity of the reactor performance, i.e. how often the washings are necessary. In general, we observe that the presence of high concentrations of organic cosolvent exerts much more drastic effects on one-point attached trypsin than in our very stabilized multipoint attached derivative. These deleterious effects of organic cosolvents are reflected in both activity and stability. In the two cases the difference found between derivatives may be mainly due to different resistance to conformational changes induced by the cosolvent. In fact, the one-point attached derivative was intrinsically more active when acting in fully aqueous medium than the multipoint attached one, either in hydrolytic or synthetic reactions. Therefore, the poorer synthetic properties found now in the synthetic behavior of this one-point attached derivative, as compared with the multipoint attached derivative, in these extreme conditions seems to be due to a lower resistance of the three-dimensional structure of the enzyme to the effect of the cosolvent.

Kinetically controlled versus thermodynamically controlled synthesis In Table 6 we compare the main parameters defining the reaction design obtained in the optimal conditions

for both kinetically controlled and equilibrium controlled synthesis. From data compared in the table, both strategies seem to offer very good possibilities for industrial scaling up when we use very active and very stable trypsin derivatives and after optimization of the design of the reaction. The main advantage of kinetically controlled synthesis is the much higher reaction rate: 5,000 versus 9 ~mol peptide per hour per milligram of immobilized trypsin obtained in equilibrium controlled synthesis. This difference is reduced to a 180-fold factor in terms of productivity of high loaded derivatives (kinetically controlled synthesis is affected by diffusional problems when using these very active derivatives). In addition to that, we have achieved experimental conditions in which the synthetic products were kinetically extremely stable and yields were approximately 100%, which represents a formidable task from a practical point of view. On the other hand, the main advantage of equilibrium controlled synthesis is the fact that now preactivation of acyl donor is not necessary. This could be very important mainly in reactions of condensation of polypeptides, and even in reactions of synthesis of dipeptides depending on the difficulty and the cost of activation of the acyl donor. In addition to that, the reaction rates per volume unit of catalyst, much smaller than in kinetically controlled synthesis, are not really too low because of the high enzyme loading (50 mg of pure trypsin per milliliter of derivative) that we have been able to reach. These overall reaction rates for equilibrium controlled synthesis seem to be also adequate to perform industrial reactions. Thus, we can calculate,

T a b l e 6 Integrated c o m p a r i s o n between different parameters in " o p t i m a l c o n d i t i o n s " for kinetically (KCS) or t h e r m o d y n a m i c a l l y (TCS) controlled synthesis of BALeuNH 2 catalysed by stabilized trypsin-agarose Strategy Initial activity mg -1 trypsin Initial activity m1-1 catalyst Linearity of time a course of reaction a Productivity b Synthetic yield in standard conditions c Synthetic yield in optimal conditions d Stability of the synthetic product Activation of acyl d o n o r Catalyst Stability e

TCS

KCS

9 / ~ m o l h -1

5,000/~mol h -1

450/~mol h 1

82,500/~mol h 1

r = 2

T = 2

1 80%

180 60%

98%

99%

Excellent

Excellent (kinetic)

Unnecessary 98

Necessary 95

a ~. factor is defined in ref. 6 b Tons of unprotected peptide per year per liter of catalyst c For 7 mM acyl d o n o r and 20 mM nucleophile For 7 mM acyl d o n o r and 200 mM nucleophile e% residual activity preserved after 2 m o n t h s of incubation in optimal conditions

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Papers from initial reaction rates and linearity of whole time courses of synthesis, a productivity of 1,000 kg of peptide per year per liter of catalyst. We have optimized the design of both strategies by synthesizing a model dipeptide. From a practical point of view, it is obvious that new economic and technological factors should be taken in account: the use of different N-protected acyl donors and C-protected nucleophiles, the price of these raw materials, solubility, downstream processes, etc. These factors could introduce important modifications in the "final compromising solution" corresponding to each synthetic strategy and they could also modify the comparison we have made between strategies.

Concluding remarks As a summary of the present paper, we would like to point out the following conclusions: (1) We have performed an integrated study of the performance of kinetically controlled synthesis of BAL e u N H 2 catalysed by stabilized trypsin. The optimal conditions for this reaction have been established by considering a number of parameters in an overall way: (i) the rate of peptide synthesis, (ii) the rate of peptide hydrolysis, (iii) the ratio rate of synthesis/rate of hydrolysis and maximum synthetic yields, and (iv) catalyst stability. (2) The optimal conditions for this synthetic reaction were pH 9.0, 4°C, 0.1 M borate buffer, 1 M ammonium sulfate. In these conditions and using equimolar concentrations of both acyl donor and nucleophile (20 mM), we have obtained the following very interesting results: Maximum synthetic yield = 54%. This yield can even be increased to more than 99% by the use of a slight excess of nucleophile in the reaction medium. 4

Initial rate of peptide synthesis = 5 mmol peptide per hour per milligram immobilized trypsin. By using our highest loaded E5 derivative containing 50 mg of trypsin per milligram of catalyst, we have obtained synthetic rates of 82.5 mmol peptide per hour per milliliter catalyst (in this case, diffusional problems induce an effectiveness factor of 0.33 with respect to the intrinsic synthetic activity of the immobilized enzyme). Considering both the initial synthetic rate and the linearity of the whole time course of synthesis, we may calculate an approximate productivity of 180 tons of unprotected dipeptide per year per liter of catalyst, which is, in our opinion, a very interesting value to work at industrial scale. The rate o f peptide hydrolysis is less than 2% corresponding to the synthetic rate, which means a very high kinetic stability of the synthetic product and hence a very simplified reactor design. This kinetic stability of the peptide product is 15-fold greater than that obtained in standard conditions, i.e. fully aqueous medium at 25°C, which are used in the most relevant papers published dealing with these type of synthesis catalysed by trypsin. 7 (3) We have performed an integrated study of the performance of equilibrium controlled synthesis of

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Enzyme Microb. Technol., 1991, vol. 13, July

BA L euN H 2 catalysed by stabilized trypsin. Optimal conditions for this reaction have been established as a "compromising solution," having in mind different parameters of industrial interest: (i) rate of synthesis, (ii) synthetic yields, and (iii) catalyst stability. (4) Optimal conditions for this synthetic reaction were pH 7.0, 25°C, 90% of organic cosolvent (2 : 1 dioxane-butanediol mixture). In these conditions and using 7 mM BA and 20 mM leuNH 2 we have obtained the following results: Synthetic yield = 80%. This yield can be increased to more than 95% by using a higher excess of nucleophile.

Initial rate of peptide synthesis = 9 I~mol peptide per hour per milligram of immobilized trypsin. This corresponds to a productivity of 1 ton of unprotected peptide per year per liter of catalyst. This value could be increased by using more concentrated agarose gels (e.g. 14%-16% gels produced by the Spanish company Hispanagar S.A., on which we have been able to immobilize up to 180 mg of pure trypsin per milliliter of support). (5) We have studied the role of some variables which are very poorly controlled in the scientific literature. These variables (e.g. polarity of the reaction medium, temperature, exact structure of the particular enzyme derivative) proved to be essential for a correct design of both synthetic approaches. (6) Stabilization of trypsin by multipoint covalent attachment has been very useful for the performance of the reaction engineering of both synthetic approaches. The use of highly stabilized enzyme derivatives has allowed us to use more drastic experimental conditions than those usually reported in the literature: (i) In kinetically controlled synthesis, we have been able to use 1 M ammonium sulfate to force the adsorption of the nucleophile on the active center of the enzyme, without promoting important conformational changes in the enzyme structure. (ii) In thermodynamically controlled synthesis, we have been able to increase the concentration of a quite apolar solvent up to 90% in order to greatly increase synthetic yields. In both synthetic approaches, our stabilized E5 derivative exhibits much better properties than nonstabilized trypsin derivatives (e.g. A1). It is much more stable, and it presents a more promising behavior as a synthetic catalyst in these more drastic optimal conditions. This very stable E5 derivative preserves more than 95% of catalytic activity after incubation for 2 months in those "optimal conditions" found for both synthetic approaches. The results from the very stable derivative E5 are very encouraging from an industrial standpoint to be used as a synthetic catalyst in the drastic optimal conditions described.

Acknowledgements We would like to thank Maria del Carmen Ceinos for excellent technical assistance. We are also very grateful to Dr. Peter Kuhl (Karl Marx Universit~it, Leipzig,

Peptide synthesis by stabilized trypsin: R. M. Blanco et al. Germany) and to Dr. Peter Halling (University of Strathclyde, Glasgow, UK) for very helpful discussions. This work has been supported by the Spanish CICYT (Project No. BIO88-0276-01). References 1 2 3 4 5 6 7

Blanco, R. M., Calvete, J. J. and Guisan, J. M. Enzyme Microb. Technol. 1989, 11, 353-359 Blanco, R. M. and Guisfin, J. M. Enzyme Microb. Technol. 1988, 10, 227-232 Blanco, R. M. and Guis~n, J. M. Enzyme Microb. Technol. 1989, 11, 360-366 Blanco, R. M., Alvaro, G. and Guise.n, J. M. (manuscripts in preparation) Kasche, V. Enzyme Microb. Technol. 1986, 8, 4-16 Fersht, A. in Enzyme Structure and Mechanism W. H. Freeman, Reading, 1977, pp. 303-312 Carleysmith, S. W., Dunnill, P. and Lilly, M. D. Biotechnol. Bioeng. 1980, 22, 753-756

8 9 10 11 12 13 14 15 16 17 18

Jakubke, H. D., Kuhl, P. and Krnnecke, A. Angew. Chem. Int. Ed. Engl. 1985, 24, 85-93 Petkov, D. D. and Stoineva, I. B. Biochem. Biophys. Res. Commun., 118, 317-323 Homandberg, G. A., Mattis, A. and Laskowski, M., Jr. Biochemistry 1978, 175, 5220-5227 Dawson, R. M. C., Elliot, D. C., Elliot, W. H. and Jones, K. M. in Data for Biochemical Research Oxford University Press, 1969 Klibanov, A. Biochem. Soc. Trans. 1983, 11, 19-43 Oka, T. and Morihara, K. J. Biochem. 1977, 82, 1055-1062 Mozhaev, V. V. and Martinek, K. Enzyme Microb. Technol. 1982, 4, 299-309 Guis~m, J. M. and Blanco, R. M. Ann. N Y A c a d . Sci. 1987, 501, 67-72 Svedas, V. K., Margolin, A. L. and Berezin, I. V. Enzyme Microb. Technol. 1980, 2, 138-144 McDougall, B., Dunnill, P. and Lilly, M. D. Enzyme Microb. Technol. 1984, 4, 114-115 Fernandez-Lafuente, R., Alvaro, G. and Guis~in, J. M. Appl. Biochem. Biotechnol. (in press)

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