Peptide synthesis by stabilized trypsin: industrial kinetic studies under extreme experimental conditions

Journal of Molecular

Catalysis, 73 (1992) 97-113

97

M2844

Peptide synthesis by stabilized trypsin: industrial kinetic studies under extreme experimental conditions Rosa M. Blancoa, Gregorio Alvaro”, Juan C. Tercerob and Jo& M. Guis6x-P “Unidad de Biocat&lisis, Institute de Catdlisis, C.S.I.C., Campus Universidad Authwmu, Cantoblanco, 28049 Madrid (Spain) bInstituto de Fisica Quimica Rocasohw, C.S.I.C., Serrano 119, Madrid (Spain) (Received August 21, 1991; accepted December 19, 1991)

Abstract Studies of the kinetics of peptide synthesis catalyzed by trypsin derivatives have been carried out. We have studied equilibrium (ECS) and kinetically (KCS) controlled synthesis using benzoyl arginine or benzoyl arglnlle ethyl ester as acyl donors and various amino acid derivatives as nucleophiles. In both cases, kinetics studies were carried out under conditions of industrial interest: (a) by using very active and very stabilized enzyme derivatives as catalysts, (b) under the extreme experimental conditions necessary for improving the performance of these synthetic processes (high concentrations of apolar organic co-solvents in ECS or high concentrations of ammonium sulfate in KCS), and (c) by using high concentrations of substrates. The hydrophobic adsorption of nucleophlle substrates on the active center of the enzyme seems to be the key kinetic step in both of these synthetic processes, as synthetic actlvlty in both ECS and KCS greatly increases with pH and with nucleophile concentration. In ECS, the presence of high concentrations of apolar organic solvents (necessary for achieving important synthetic yields) greatly inhibits the adsorption of nucleophiles, and hence the best performance of these processes would be obtained using a moderate excess of the nucleophile substrate in order to quantitatively convert the acyl donor substrate into synthetic product at the highest reaction rates. In KCS, we found that the use of a ‘salting out’ agent (e.g., ammonium sulfate) greatly improves the hydrophobic adsorption of nucleophiles. In addition, the presence of ammonium sulfate, perhaps as a ‘water-ordering agent’, promotes a dramatic reduction in the rate of hydrolysis of the acyl-enzyme complex when the active center of each trypsin molecule is partlly blocked by the adsorbed nucleophlle at saturating concentrations. Thus synthetic/hydrolytic ratios are dramatically improved by the presence of this salt, and consequently synthetic yields approach 100%. By using these two different synthetic strategies we have been able to quantitatively convert arginyl derivatives into dipeptides under extreme conditions in which our stabilized trypsin derivatives are very active and very stable. We have calculated catalyst productivities of 2 and 300 tons of dipeptide per year per liter of our trypsin catalysts by using ECS or KCS synthetic strategies, respectively.

Introduction Kinetics studies of the behavior of industrial enzyme derivatives under experimental conditions of industrial interest (iridustrial enzyme kinetics) 0304~6102/92/$5.00

0 1992 - Elsevier Sequoia. All rights reserved

98

may constitute a ‘key point’ with which to evaluate and improve the practical possibilities of scaling an enzymatic process up to an industrial level. These kinetics studies should include: (i) enzyme reactions of real practical interest, or very similar ones; (ii) high substrate and product concentrations, similar to those used during the industrial performance of the enzyme processes; (iii) experimental conditions of practical interest (e.g., non-conventional media); and (iv) the use of immobilized-stabilized derivatives as potential industrial catalysts. In this way, these kinetics studies would allow us to directly establish the range of experimental conditions (e.g., pH value, substrate concentrations) under which to perform these industrial processes. In addition, these kinetics studies may also provide important information on the enzyme mechanism under these extreme experimental conditions of real practical interest. Obviously, these enzyme mechanisms and their main features (for example, distorting or inhibitory effects promoted by high substrate, product, or solvent concentrations) may be quite different from those indicated by classical kinetics studies performed on model reactions of no practical interest catalyzed by immobilized enzymes. Hence, industrial enzyme kinetics should provide the correct information for establishing the ‘rational basis’ on which to improve the technological performance of industrial enzymes, through protein design of improved soluble enzymes, through chemical modification of immobilized derivatives, and so on. Peptide synthesis catalyzed by proteases is a good example of an interesting industrial enzyme process. This enzymatic approach offers several important advantages over more classical and conventional chemical methods: no risk of racemization, the absence of side reactions, the use of milder experimental conditions, and so on [ 11. However, many important drawbacks must be overcome when we try to progress from discovering these interesting processes (e.g., peptide synthesis catalyzed by trypsin) to the scaling up to industrial levels. We have prepared very active and very stable trypsin derivatives by means of an original strategy for immobilization-stabilization of enzymes by multipoint covalent attachment to agarose-aldehyde gels 121. These trypsin derivatives contain up to 100 mg of pure enzyme per ml of catalyst, and they exhibit very high levels of activity (70% of the activity corresponding to soluble enzyme) and stability (they are 12 OOO-fold more thermostable than soluble enzyme in the absence of autolysis phenomena). These derivatives were attached to agarose supports through 7 covalent bonds per immobilized molecule [ 31, and they also proved very stable against a number of denaturing agents (urea, organic co-solvents, and so on). For all these reasons, these trypsin derivatives seem to be well suited for use as industrial catalysts in synthetic processes. We have also reported a first approach to the design of peptide synthesis by these stabilized trypsin derivatives. We have tested the integrated effect of different variables that define the reaction medium on various parameters of industrial interest. Optimal conditions (the nature and concentration of

99

organic co-solvents, ionic strength, temperature, range of pH values) for performing the industrial synthesis of peptide bonds were dellned. These experimental conditions were established as ‘compromise solutions’ that allow us to obtain good values of very different scale-up parameters (such as activity and stability of the catalyst, synthetic yields, stability of the synthetic product, and so on) simultaneously. The use of very stabilized trypsin derivatives instead of non-stabilized ones allowed us to greatly improve the design of these synthetic processes, because we were able to use experimental conditions quite a bit more extreme than those allowed when using nonstabilized trypsin derivatives [4]. In this paper we present a study of the kinetic behavior of these stabilized trypsin derivatives as catalysts of peptide bond synthesis under extreme experimental conditions. We have studied the synthesis of various dipeptides of arginine by means of two different approaches: equilibrium controlled synthesis (direct condensation of benzoyl arginine, BA, with various amino acid amides in the presence of high concentrations of organic co-solvents) and kinetically controlled synthesis (benzoyl arginiie ethyl ester, BABE, used as an activated acyl donor in the presence of high concentrations of ammonium sulfate). In each case, we have tried to obtain information on the mechanism of enzyme action under these extreme experimental conditions, as well as to define the exact reaction conditions (e.g., pH value, substrate concentrations) under which can be obtained the best values of catalytic activity during the whole time-course of these industrial synthetic processes. Of course, not to neglect the practical point of view, synthetic yields and easy product recovery were also considered.

Experimental

Crosslinked 10% agarose gels were donated by Hispanagar (Burgos, Spain). The enzyme trypsin (E.C.3.4.21.4) from bovine pancreas, BABE (benzoyl arginine ethyl ester), BA (benzoyl arginine), LeuNHz (leucinamide), TyrNHz (tyrosinamide), and GlyNHz (glycinamide) were purchased from Sigma, 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 BABE units per milligram of protein. Preparation of trgpsin ~aw&..e+agarose (al&h&e) ciimivative Multipoint covalent attached trypsin-agarose derivative was prepared as previously described [ 31. The main characteristics of this derivative are: activity= 70%, corresponding to soluble enzyme that has been immobilized; stabilization= 12 000, defined as the ratio of the half-lie of each derivative to the half-life of soluble enzyme in the absence of autolysis phenomena; enzyme loading: 100 mg enzyme per ml of gel. In some cases we also used a very low-loaded trypsin derivative (0.5 mg of pure enzyme per ml of packed

100

catalyst) in order to prevent diffusional limitations that may innuence the apparent behavior of the immobilized enzyme. Synthetic reactions Kinetically controlled sgtiheti This reaction was performed in a small beaker very gently stirred inside a high-low temperature incubator. The assay mixture was made from 10 ml of 0.1 M borate buffer at various pH values, with various concentrations of the reactants BAEE and various nucleophiles. The reaction was started by adding 350 mg of wet low-loaded derivative (equivalent to 0.5 ml of packed gel and hence to 0.25 mg of immobilized trypsin). The reactions were performed at 4 “C. In some cases the effect of the presence of 1 M ammonium sulfate in the reaction mixture was also studied. Thermodgnumicall~ controlled sgtih.esi.s The reactor was a thermostated jacket column (25 “C) packed with 10 ml of our highly loaded trypsin-agarose derivative. Before starting the reaction, the cohunn was equilibrated by fluxing 50 ml of the water-organic co-solvent mixture. The reaction mixture was composed of 100 ml of 7 n&I BA and various concentrations of nucleophiles dissolved in a mixture of dioxane-butanediol-water (6:3:1) at various pH values. No buffer was used, because the nucleophiles act as buffers in the range of pH values studied. For kinetic analysis, the reaction mixture was pumped through the column at various flow rates (from 0.1 to 2 ml min-‘), and aliquots of the eluted solution were analyzed by HPLC. Reaction rates were obtained from plots of percent conversion 2)s. residence times. For equilibrium analysis, the reaction mixture was fluxed through the column at a constant flow rate (0.7 ml min-‘), and the first 50 ml eluted out from the column were discarded. The reaction mixture was then recirculated through the column at the same flow rate, with continuous external adjustment of pH using 1 M NaOH as titrant. Corresponding amounts of co-solvent were also added, in order to maintain a constant co-solvent concentration. The use of highly concentrated t&rant solution prevented excessive dilution of the reaction mixture (the volume was increased less than 5%), and so dilution of reactants was minimal. HPLC anal&s Substrates (BA or BAEE) and products (peptides, and also BA in the kinetically controlled synthesis) were separated and qua&tied by reversephase HPLC. At various intervals aliquots of the supernatants were withdrawn and diluted with 4 volumes of mobile phase, filtered through a Millipore titer (0.45 q), and injected. A Konic Instruments (San Cugat, Spain) with a Spectra Physics SP 8450 UV detector and a 250X4.6 mm RP-C 18 (5 pm) column (Spherisorb@) was used. Samples were eluted isocratically with a mixture of 40% ethanol, 60% water containing 0.1% phosphoric acid as the

101

mobile phase in the kinetically controlled experiments, and 50% ethanol, 50% water containing 0.1% phosphoric acid in the thermodynamically controlled synthesis. The flow rate was 1 ml min- ‘, and the amounts of reactant and products were determined from the calibration curves using stock solutions. Results and discussion

Equilibriumcmtrolled

s@hesG

(Except where specified otherwise, we studied the direct condensation of benzoyl arginine and leucinamide in the presence of 90% of an organic solvent (a 2:l mixture of dioxane and butanediol) to yield the dipeptide benzoyl arginine leucinamide.)

Eflect of pH In Fig. 1 we compare the very different patterns for yields-pH and activity-pa. On the one hand, synthetic yields are maximal and practically constant within a fairly wide range of pH values (between 5.8 and 7.2). The concentrations of non-ionized forms of the acyl donor and the nucleophile are influenced by pH in opposite ways: when the pH increases, the nonionic form of the nucleophile (amine group) increases, while the non-ionic form of the acyl donor (carboxyl group) decreases. Thus it can be assumed that there is a range of pH values in which the product of the concentrations of the non-ionized forms of the reactants remains constant. This product is precisely the one that defines the equilibrium position [ 51, and hence the maximal synthetic yields must be fairly independent of pH within this wide range of values. Interestingly, these results, which agree with thermodynamic 10

100

3 75

3 iE

E

co50

z?

z .-Q,

d>

)I 25 2

a

7

6

PH

D

F’ig. 1. Effect of pH on yields and initial rates of the thermodynamically controlled synthesis reaction of E&-Leu-NH2. (B): yields given in percent conversion of acyl donor (HA); (0): initial rates, in mM peptide h-’ (mg enzyme)-’ X 10V3. Leucinamide concentration= 20 mM, temp. 25 “C.

102

predictions, allow us to test the kinetic behavior of our derivatives in this wide range of pH values in which synthetic yields are maximal. On the other hand, the rate of synthesis dramatically increases with increasing pH (Fig. l), paralleling the increase in the concentration of the non-ionic form of the amine nucleophile. The simultaneous decrease in the concentration of the non-ionic form of the acyl donor substrate does not seem to have a strong influence on the overall reaction rates, although it might greatly reduce the rate of formation of the acyl-enzyme (BA-trypsin) complex. This result suggests that the controlling step of this synthetic process, under these experimental conditions, may be the nucleophilic attack of the non-ionic form of the nucleophile (leucinamide adsorbed on the active center of the enzyme) on the acyl-enzyme covalent complex. So, the increase in pH, increasing the concentration of non-ionized leucinamide, may greatly improve the hydrophobic adsorption of leucinamide on the active center of the enzyme, which must be strongly inhibited by the presence of very high concentrations of organic co-solvents [ 4 1. From a technological point of view, the performance of these synthetic processes at pH 7.0 indicates that this is the best solution for obtaining good activity and equilibrium values simultaneously. Eflect of substrate concentrations The concentration of acyl donor, between 0.5 and 10 mM, hardly affects the synthetic rates (not shown), but synthetic activity does greatly increase with increasing nucleophile concentration (Table 1). These results agree with assumptions made in the previous paragraph. The nucleophilic attack of free leucinamide adsorbed on the active center of trypsin towards the acyl-enzyme complex, BA-trypsin, seems to be the controlling step of these synthetic processes under these extreme experimental conditions. Usually, obtaining quantitative yields (Le., higher than 95%) when using equimolar concentrations of both substrates in these condensation processes is extremely difficult. From a practical point of view, in this type of process an excess of one of the two substrates should be used, in order to achieve quantitative conversions (Le., higher than 95%) of the other one. In this TABLE 1 Effect of the concentration of the nucleophile in the thermodynamically controlled synthesisa mM Leucinamide

VOb Yields’

7

20

100

200

7.8 60

9 80.7

13 95

15.5 97

Temp.

25 “C; pH 7.0. . bInitid rates in mM peptide h-’ (mg enzyme)-‘x

Welds:

percent conversion of acyl donor.

10w3.

103

way, further product recovery processes would be simplified: we would have to separate a condensation product from an excess of only one substrate. The selection of the substrate to be used in excess is thus a technological ‘key point’, and kinetics studies (like the ones mentioned above) should provide the first and perhaps the best selection criteria. In our case, then, the use of an excess of nucleophile, e.g., leucinamide, in order to quantitatively convert arginine derivatives into dipeptides seems to be the option of choice. Of course, in some cases, other factors, such as cost, solubility and stability of the substrates, and so on, may have to be considered. In Table 1 we also show the important increase in the synthetic yield (with respect to the acyl donor, BA) when we increase the concentration of the nucleophile leucinamide. When using 200 mM leucinamide, however, the yields obtained by changing the acyl donor concentration, from 0.5 to 10 mM, remain unaltered (data not shown). These results clearly agree with thermodynamical predictions. From the equilibrium equation of the reaction:

Kt,hlUW=

[peptide 1

Knon_ion[ acid 1[amine 1

the thermodynamic yield will be: [peptide] =K*K,,,i,,[acid][amine]/a, Since we are using an excess of nucleophile agent, we can assume that [amine] is approximately [amine]O. In addition, we can also establish the following mass balance: [acid],, = [peptide] + [acid] Then yield = [peptide]/[acid],

=

K[~elo+a, K[aminel,

where K = Ka Knon_ione From this equation, we can conclude that when using an excess of nucleophile agent, the synthetic yields with respect to the acyl donor substrate must be independent of the acyl donor concentration, and they must obey a saturating dependence on nucleophile concentration. This is exactly what we have observed experimentally. Thus we can assume that synthetic yields are really equilibrium controlled ones, and they are not restricted by additional kinetic factors (e.g., strong product inhibition). In this way, we are indeed able to quantitatively convert the acyl donor substrate into dipeptide product (for instance, 97% yield is obtained with 200 mM leucinamide). We would also like to remark that the whole course of these synthetic processes is fairly linear. For example, the time required to achieve 90% conversion (from 7 mM BA and 200 mM leucinamide) is less than 50% higher than the extrapolated time if the reaction rate were always constant and equal to the initial reaction rate. Again, these results clearly indicate

104

the absence of product inhibition (even a fairly weak one) during the whole course of synthesis. Nature of the nucleophik agent Three amino acid amides were tested as nucleophiles for this synthetic reaction: leucinamide, tyrosinamide and glycinamide. The yields of dipeptides obtained with the same acyl donor (BA) and these different nucleophiles were essentially identical (Table 2). These results are logical, given the thermodynamic predictions. The pK values for these three amino acid amides are almost identical, and the intrinsic equilibrium constants for the formation of these three similar peptide bonds must also be very similar. These results also indicate that we are able to get quantitative conversions (Le., higher than 95%) of BA into different dipeptides by using moderately high concentrations of amino acid amides. Nevertheless, the rates of these three reactions seemed to be strongly affected by the nature of the nucleophile agent used, and especially with its polarity. As shown in Table 2, the reaction is faster with less polar nucleophiles. Trypsin preferentially accepts hydrophobic derivatives of amino acids as nucleophiles. This specificity results from the predominantly hydrophobic character of the subsite of the leaving group in its active center. The kinetic behavior of the reaction will be affected by this hydrophobic specificity, and thus higher rates were achieved with leucinamide and tyrosmamide as nucleophiles. The slowest reaction was the one performed with glycinamide, which is less hydrophobic and whose adsorption on this subsite is correspondingly more difficult. Free leucine was also tested, but it could not be recognized as a nucleophile by the enzyme, and no synthesis occurred at all. The amino acid without N-protection is probably in the anionic form at the reaction pH. Thus it must be repelled by the hydrophobic subsite,where the existence of a negative charge has been described [6]. This fact must be closely related to the endoprotease character of trypsin: repulsion toward carboxy terminal groups makes it unrecognizable as a substrate, whereas it is recognizable when esterified or part of an amide or peptide bond.

TABLE 2 Effect of the nature of the nucleophile in the thermodynamically controlled synthesis”

vo b Yields’

Leucinamide

Tyrosinamide

Glycinamide

9 80

7 79

1.5 78

“Concentration of nucleophiles=20 mM; pH 7.0; temp. 25 “C. %itial rates in mM peptide h-’ (mg enzyme)-’ X 10m3. ‘Yields: percent conversion of acyl donor.

105

Kinetically controlled synthesis (Unless spectied otherwise, the reaction studied was the synthesis of BA-LeuNH2 from benzoyl argmine ethyl ester and leucinamide.) Eflect of pH Synthetic yields and reaction rates obtained at various pH values are shown in Rig. 2. Reaction rates behave as follows: the rate of acid (BA) formation remains practically constant over this range of pH values, whereas the rate of peptide synthesis greatly increases with pH, and the rate of peptide hydrolysis is similar throughout the pH range. Because of the ratios between these different activity values, the synthetic yields greatly increase with pH. The shape of the curve in F’ig. 2 looks very much like a titration pattern corresponding to the amino group of leucinamide, and gives a pK of 7.8 [7]. This means that the degree of ionization of leucinamide molecules and consequently the increase in the percentage of trypsin molecules containing free leucinamide adsorbed on their active centers seems to be the only factor responsible for this parallel increase in synthetic rates and in yields. If other factors were also very important (for instance, if the capacity of this trypsin derivative to adsorb leucinamide changed with pH), then we should observe a much more complex pattern - the synthetic rates at pH 9.0 or 10.0 would be different, and/or the apparent pKs obtained from Rig. 2 would be very different from the value found for leucinamide. From a practical point of view, pH 9.0 seems to be the most suitable pH at which to run these syntheses: the highest synthetic yield and rate are achieved at either pH 9.0 or 10.0, but at pH 9.0 the stability of the enzyme is higher and the spontaneous hydrolysis of the ester is negligible.

a

0

10

PH Fig. 2. Effect of pH on maximum yields of the kinetically controlled synthesis reaction of BA-La-NH2. Yields: percent conversion of acyl donor (EMEE). Concentration of EUEE and of leucinamide = 20 r&L Aqueous medium. Temp. 25 “C.

106

Effect of concentration of substrates As with previously reported results [4], these kinetic studies were performed under two very different sets of experimental conditions: standard conditions (fully aqueous medium) and extreme conditions (presence of 1 M ammonium sulfate). A& donor CBA). Concentrations between 20 and 200 mM were tested, while maintaining a constant concentration of nucleophile (0.2 M). Initial rates of synthesis and hydrolysis obtained under the two different experimental conditions remained constant in each case, in the entire range of substrate concentrations, so it may be assumed that these are saturating concentrations of the acyl donor. Nuclmphile (Zeucinumide). The nucleophile concentration is a ‘key variable’ in the kinetics of these complex synthetic processes [8]. We can represent the mechanism of kinetically controlled synthesis schematically as follows:

BAEE

+ LeUNHZ

+ Hz0

Pansferase )

(S) BA-LeuNH2

+ EtOH

+ Hz0

(Hz) Hydrolase BA + EtOH

+ LeU-NH,

The exact kinetic mechanism of this complex process has been discussed thoroughly by Kasche [ 71 and by Konecky [8]. We can now establish some simple assumptions related mainly to the practical performance of these synthetic processes: (i) In the first stages of the synthetic process, the percentage of BAEE molecules that are converted into dipeptide is closely related to the ratio between the rate of aminolysis and the rate of hydrolysisl. This ratio will also define the maximal synthetic yield when the rate of peptide hydrolysis (hydrolysis,) is quite lower than the rate of synthesis (aminolysis) or when we are able to continuously remove the peptide product. (ii) Aminolysis of the enzyme-acyl donor complex occurs only through nucleophilic attack of the nucleophile adsorbed on the active center of the enzyme [7]. ln fact, the aminolysis/hydrolysis ratios usually reported in the literature depend strongly on the enzyme specScity toward different nucleophiles [91. In addition, the saturating curves usually found for the rateof-aminolysis-nucleophile patterns also support this assumption [ 10 I. (iii) We can consider a simple critical event for this synthetic process - the deacylation of the BA-trypsin complex that is formed during the reaction of the activated acyl donor, BAEE, with the immobilized enzyme. At this moment we can consider two kinetic possibilities:

107 HZ BA-LeuNH,.,

jfuNH%BAEE

+ LeuNH2

,

BA

BA

+ LeuNH

+

LeuNH,

2

+ E

SL BA*E

H

+ LeuNH,

t--

l,o BA

+ LeuNH2

(a) The acyl-enzyme complex is formed on trypsin molecules that did not or do not have nucleophile adsorbed on their active center. In this case the deacylation process will only be hydrolysis of the BA-trypsin complex (hydrolysisl,o), and this rate of hydrolysis will be exactly the same as that found for the hydrolysis of activated acyl donor (e.g., hydrolysis of BABE by trypsin) in the absence of nucleophile. (b) The deacylation process occurs in trypsin molecules that have nucleophile adsorbed on their active center. In this case deacylation may occur via two different mechanisms: hydrolysis of the BA-trypsin complex in the presence of leucinamide (hydrolysis,,i) or aminolysis of the BA-trypsin complex by nucleophilic attack performed by the adsorbed leucinamide (S). Of course, hydrolysis,,, may be quite different from hydrolysis,,o because of different mechanistic possibilities: when the controlling step of the overall process is the acylation of the enzyme, the nucleophile will compete with water in the fastest deacylation process; when the controlling step of the overall process is the deacylation step, the presence of nucleophile adsorbed on the active center of the enzyme may exert some positive additive effects on the deacylation ratio, but it may simultaneously also hinder the accessibility of water to hydrolyze the acyl-enzyme complex. Even from these simple assumptions it is evident that the activities-nucleophile patterns may reflect a large number of very different mechanistic events. The rate-of-synthesis-nucleophile patterns could be more easily discussed by considering a simple theoretical equation: Vw,,th,,,[leucinamide] V synth = [ leucinamide] + Kads,-ieu

where Vsynth,max is the maximal rate of synthesis, obtained when every trypsin

molecule has a nucleophile molecule adsorbed on its active center when the BA-trypsin complex is starting to deacylate, and Kads represents an overall adsorption constant between trypsin and leucinamide. Of course, as discussed by Kasche, this adsorption may be quite different for the different microscopic processes, e.g., adsorption of leucinamide on free trypsin, adsorption of leucinamide on BA-trypsin complex, etc. [7]. On the other hand, the rateof-hydrolysis-leucinamide pattern and the rate-of-BAEE-consumptionleucinamide pattern would be even more complicated, and this will be discussed only in a very qualitative way.

108

We have tested the effect of nucleophiie concentration, varying between 20 and 200 mM, on the rate of synthesis (formation of the dipeptide BA-leucinamide) and on the overall rate of hydrolysis, (formation of BA) when using 20 mM of activated acyl donor BABE under the two different experimental conditions (standard and extreme) described above. Under both conditions, the synthetic rates greatly increase with nucleophile concentration, following fairly saturating curves (Fig. 3). In fact, reciprocal plots (1 /synthetic rate VS. l/[leucinamide]) are fairly linear (F’ig. 4) and allow us to calculate 1.251

1.25

0.25

0’

(al

0

?a0

IeuGamide FM)

0

leucinamide (mM)

Fig. 3. Effect of the concentration of nucleophile on the kinetically controlled synthesis of BA-Leu-NH2: (a) reaction performed in aqueous buffered solution; (b) reaction performed in 1 M ammonium sulfate. (m): rate of decomposition of BAEE; (0): rate of formation of BA; (A): rate of formation of dipeptide. Reaction temp. 4 “C, pH 9.0. Concentration of BAEE: 20 mM.

Fig. 4. Lineweaver-Burk plots for the kinetically controlled synthesis reaction of BA-Leu-NH$ effect of the presence of 1 M ammonium sulfate. Ordinate: (initial synthesis rates)-’ (rate units: mM min-‘); abscissa: (leuchamide concentration)-’ (concentration units: mM). (0): reactions in aqueous buffered solution; (m): reactions in 1 M ammonium sulfate. All reactions performed at pH 9.0, 4 “C. Concentration of WE: 20 mM.

the maximal synthetic rates (1.07 and 1.37 mM min- ‘) and the overall adsorption constants (34 and 123 mM) in the presence and absence of ammonium sulfate, respectively. Thus the presence of ammonium sulfate seems to improve the hydrophobic adsorption of free leucinamide on the active center of trypsin, but it does not exert important effects on the maximal synthetic rates. In contrast, the hydrolytic rate (hydrolysis, = (overall formation of benzoyl arginine instead of peptide) = hydrolysis,,0x hydrolysis,,,) decreases when the nucleophile concentration does, and the hydrolysis-nucleophile patterns in the absence and in the presence of ammonium sulfate are very different. Under standard conditions the hydrolytic rates decrease slightly when the nucleophile concentration increases, and they asymptotically approach a constant but still significant value, which may represent the relative value corresponding to the nucleophilic attack of water on the BA-trypsin (Vhydr0lySisl,) complex h the presence of nucleophile adsorbed on the enzyme active center. This value is approximately 40% of the value for hydrolysis in the absence of nucleophile.(V,,.,~olysisl,l = 0.4 X Vb*obsisl,J, and this might indicate that the presence of nucleophile adsorbed on the active center of the enzyme presents some steric hindrance to the nucleophilic attack of water molecules. However, in the presence of ammonium sulfate, hydrolytic rates strongly decrease with nucleophile concentration, and they trend to zero 0). This dramatic reduction in the rate of hydrolysis,,, could (Vhydr0lySis1.1= also be explained by the steric hindrance promoted by the adsorbed nucleophile. Now, the presence of this important lyotropic salt (Hofmeister lyotropic series) greatly increases the degree of structural organization of water molecules (entropy of water decreases) [ 111. Hence the accessibility of these highly structured water molecules to the acyl-enzyme complex, partially blocked by the presence of the adsorbed nucleophile, may be strongly inhibited, and this would explain the dramatic reduction in hydrolytic activity. Of course, the presence of this high concentration of ammonium sulfate might also promote some distortions of the trypsin structure. However, these distortions do not seem to be dramatic enough to cause such very interesting mechanistic changes. In fact, synthetic rates in the presence and absence of salt are practically identical, and rates of BAEE hydrolysis are only moderately different. Thus the mechanism of action of ammonium sulfate described above seems to be quite logical, and it may be extremely interesting as a powerful general tool for improving the industrial performance of these complex kinetically controlled syntheses. In every case in which there is some experimental evidence of hindrance to the accessibility of water due to the nucleophile adsorbed on the enzyme active center, we would propose the use of lyotropic salts or other water-ordering agents (polyols, sugars, and so on) to greatly reduce hydrolytic activity (hydrolysis,,,) and hence to greatly increase the aminolytic/hydrolytic ratio and the synthetic yields. In fact, in the present case we can observe (Fig. 5) that the presence of ammonium sulfate causes a dramatic increase in the synthesis/hydrolysis ratio and hence in synthetic yields. Evidently this increase in s/h ratio is

200 (4

leucinamide (mM)

l&cinaze

(ml;)

Fig. 5. Effect of the presence of ammonium sulfate in the kinetically controlled synthesis of BA-Leu-NH,: (a) ratio of synthesis/hydrolysis rates; (b) maximum yields (percent conversion of acyl donor BAEE). (0): reactions in aqueous buffered solutions; (I): reactions in 1 M ammonium sulfate. All reactions performed at pH 9.0, 4 “C. Concentration of BAEE: 20 mM.

not due to an increase in synthesis rates but rather to a sharp decrease in the hydrolysis rate, as noted above. In addition, Hyun et al. have also reported very interesting increases in kinetically controlled cephalexin synthesis catalyzed by an acylase from X. citri promoted by the presence of high concentrations of sorbitol [ 121. These increases were due mainly to a decrease in the hydrolytic rate, as we have reported here, and we could explain these results by a very similar mechanism. The same effect applies in the hydrolysis of the peptide H2. Our reaction courses in the absence of ammonium sulfate as well as most of those reported for kinetically controlled reactions catalyzed by trypsin show a bell shape. This means that for practical application of the processes, extremely careful control is required in order to obtain the condensation product before it is hydrolyzed. Some methods have been described for solving this problem, such as precipitation of the products when soluble enzymes are used as catalysts, or the use of biphasic systems in which enzyme and product are in separated phases. When our reaction was performed under optimal conditions of nucleophile concentration and in the presence of 1 M ammonium sulfate, this hydrolysis of H2 was also very much diminished. Moreover, the reaction is fairly linear, which indicates that there is no inhibition by the product. Thus the reaction course in these conditions has a ‘saturation’ shape: maximum yields are kept constant. This fact makes the process easier to control, which is extraordinarily important when scale-up is a goal. Our results also compare favorably with other studies reported in the literature. For example, Oka and Morihara reported a value of kS/k,= 800 for the same synthetic reaction (synthesis of BA-leucinamide from BAEE and leucinamide catalyzed by soluble trypsin in fully aqueous media at pH 10.4 and 25 “C) [9]. We were able to increase this value up to 5 100 by using immobilized-stabilized trypsin derivatives and very different experi-

111

mental conditions (i.e., 1 M ammonium sulfate, pH 9.0, and 4 “C). In fact, this very high value is also quite a bit higher than most listed by Kasche for a number of synthetic enzymes with industrial interest [ 71. This comparative kinetic parameter was calculated according to Kasche’s equation: where V,, is the initial rate of peptide formation, VAOHis the initial hydrolysis rate, and [NH] is the molar concentration of nucleophile. Finally, from a technological point of view, we can conclude that under extreme experimental conditions (e.g., in the presence of 1 M ammonium sulfate) the performance of these synthetic processes is greatly improved: (i) adsorption of nucleophile is improved and hence lower concentrations can be used while stiIl achieving maximal synthetic yields; (ii) the rate of peptide hydrolysis is dished and hence the peptide product becomes very stable; (iii) at high nucleophile concentrations we are able to get extremely high aminolysis/hydrolysis ratios and hence quantitative conversions (around 100%) of activated acyl donor, BAEE, into the peptide product, and hence downstream processes to recover the dipeptide become greatly simplilled; (iv) as previously reported, our multipoint covalent attached trypsm derivatives were exceptionally stable under these extreme experimental conditions, much more stable than soluble or one-point attached trypsin derivatives; (v) in this way we can obtain a very high synthetic productivity for quantitative conversions (more than 99%) of 50 mM of BAKE in the dipeptide BA-leucinamide. Nature of the nucleophile agent Various peptides were synthesized using tyrosinamide, glycinamide, and leucine as nucleophiles, maintaining BAEE as acyl donor, and the results were compared with the ones obtained using leucinamide (Table 3). When the nucleophile is rather hydrophobic, as are leucinamide and tyrosinamide, yields are high and very close in value. But when the nucleophile is glycinamide (much less hydrophobic than the former) the yields are very poor. This may mean that achieving adsorption with this hardly hydrophobic molecule on TABLE 3 Effect of the nature of the nucleophile in the kinetically controlled synthesis” Maximum yieldsb

standard conditionsc optimal conditionsd

Leuchwnide

‘&-rosinamide

Glycinamide

32 54

42 60

6 6

‘Concentration of nucleophiles and BAJSE= 20 mM. bMaximumyields: percent conversion of acyl donor. ‘0.1 M Borate buffer, pH 9.0, 25 “C. dl M Ammonium sulfate in 0.1 M borate buffer, pH 9.0, 5 “C.

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the active center of trypsin must be much more difficult than with tyrosinamide or leucinamide. As a result, the nucleophile attack occurs in much lower proportions, and deacylation of the acyl-enzyme complex must be carried out predominantly by water. Thus the kinetics are very slow and the yields very poor. Free leucine was also tested as a nucleophile, but the enzyme does not recognize it at pH 9 or 10. This suggests once more that the lack of recognition must be due not to the different pK values of leucine and leucinamide, but rather to the negative charge of leucine preventing its adsorption on the hydrophobic subsite of the active center of trypsin. This repulsion cannot be avoided, even when using very high ionic strength.

Conclusions

Kinetics studies of the behavior of immobilized-stabilized trypsin derivatives as catalysts of synthetic processes under extreme experimental conditions have provided us with the ‘rational basis’ with which to understand and perform these important industrial processes. Interestingly, the adsorption of the nucleophile amino acid derivative (e.g., leucinamide) on the enzyme active center seems to be the ‘key point’ in these reaction mechanisms (kinetically and equilibrium controlled synthesis). From this point of view, classical kinetics studies performed with trypsin derivatives on model reactions (e.g., hydrolysis of model amino acid esters in the absence of nucleophile) may not be very relevant for testing the synthetic possibilities of these derivatives. We ourselves chose the organic solvent in which to perform equilibrium controlled synthesis from these types of model studies. Now, as a result of studies presented here, we will have to test different co-solvents in real synthetic processes in order to find the ones with which are obtained the strongest adsorption of the nucleophile and the best substrate solubility (for different arginine derivatives). From the industrial kinetics studies presented here, we can conclude that the use of an excess of nucleophile ammo acid at pH 7.0 seems to be the option of choice when performing equilibrium controlled synthesis of arginine or lysine derivatives catalyzed by stabilized trypsin. So, by using 200 mM of leucinamide, tyrosinamide, or glycinamide we will be able to get quantitative conversions (around 98%) of high concentrations of arginyl derivative (e.g., 50 mM). In this case, we have been able to work only with 10 mM of benzoyl arginine. However, the solubilities of various arginyl and lysil derivatives in various solvents are now being studied in our laboratory. By using our high loaded trypsin derivatives (100 mg of pure trypsin per ml of catalyst) we are able to get important peptide productivities under these extreme experimental conditions - for example, 2 tons of arginine-leucinamide per year per liter of catalyst. The presence of 1 M ammonium sulfate greatly improves the performance of kinetically controlled synthesis of peptide derivatives. The presence of

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this salt promotes a better adsorption of the nucleophile amino acid, a slower peptide hydrolysis, and a dramatic decrease in the hydrolysis of the acyl-enzyme complex in the presence of adsorbed nucleophile. In this way we have been able to get quantitative conversions (around 100%) of 50 mM of BABE in dipeptide by using a moderate excess of nucleophile amino acid (e.g., leucinamide). By using our high loaded trypsin derivatives (100 mg of pure trypsin per ml of catalyst) we are able to get important peptide productivities under these extreme experimental conditions - for example, 300 tons of arginint+leucinamide per year per liter of catalyst. We have proposed an interesting kinetic mechanism to explain the effect of ammonium sulfate on the reduction of hydrolytic activities during kinetically controlled synthesis. This mechanism may have general applicability for improving the industrial performance of these complex synthetic processes. Ammonium sulfate, or other water-ordering agents (lyotropic salts, sugars, polyols, etc.), increase the degree of organization of water molecules and dramatically reduce their ability to act as nucleophile agents when the active center of the enzyme is partially blocked by the presence of adsorbed nucleophiles. In this way, synthetic yields and the aminolysis/hydrolysisratio that defines them will increase dramatically, because of the reduction (down to zero) of the hydrolytic activity, and not because of any dramatic increase in the aminolytic rates, which may actually be impossible.

Acknowledgements We would like to thank Rafael Armisen (Hispanagar, Burgos, Spain) for his generous gift of 10% agarose gels and M. Carmen Ceinos for skilled technical assistance. This work was supported by Spanish CYCIT, Project No. BI088-0276-01.

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