(My apologies for some missing features in this web version - something in the file supplied defeated my repeated attempts at conversion to HTML, so I eventually had to do this indirectly via plain text - Peter Halling)
THE EFFECT OF CROWN ETHERS ON THE ACTIVITY OF ENZYMES IN ORGANIC SOLVENTS.
Dirk-Jan van Unen, Johan F.J. Engbersen, and David N. Reinhoudt
Laboratory of Supramolecular Chemistry and Technology
University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands
Tel. + 31 53 4892980; Fax + 31 53 4894645; E-mail: SMCT@ct.utwente.nl
1. Introduction
Nowadays the applicability of enzymes in synthetic organic chemistry is well recognized. The field of enzyme-catalyzed organic synthesis has been further boosted by the recognition that enzymes can operate in organic solvents. The use of non-aqueous media for enzymatic conversions offers a number of advantages, like enhanced thermal stability of the enzyme, increased substrate solubility, a shift of the equilibrium in favor of synthesis over hydrolysis, and altered selectivity properties of the enzyme.[ref] However, the most important drawback of the use of enzymes in organic media is the reduced catalytic activity compared to aqueous conditions. Typically, this reduction in activity is two to six orders of magnitude.[ref] However, studies in our laboratory have revealed that (pre)treatment of the enzymes with crown ethers can enhance enzyme activities in non-aqueous organic solvents up to a level approaching the activity under aqueous conditions.
Crown ethers, discovered in 1967 by Pedersen[ref], are cyclic polyethers composed of ethylene-oxy units. They have an extraordinary ability to solvate alkali metal ions by sequestering the metal in the center of the polyether cavity. The selectivity for cation complexation is dependent on the size of the ring, e.g. 18-crown-6 [2] is selective for the K+ ion whereas the smaller 15-crown-5 [1] is selective for the Na+ ion. Furthermore, 18-crown-6 is able to complex ammonium ions[ref] and water.[ref] The selectivity of crown ethers cannot only be tuned by variation of the ring size, also the number and type of donor atoms can be altered, an example being monoaza-18-crown-6 [3]. Substituents on the ring, like in dicyclohexyl-18-crown-6 [4], may change the flexibility and geometry of the ring, as well as the electronegativity of the donor atoms. Upon introduction of an additional bridge the name is changed from crown ether into cryptand, like kryptofix 2.2.2 [5].
INSERT CHART CROWN ETHER STRUCTURES
Interactions between proteins and crown ethers could be expected to occur since proteins possess lysine ammonium groups. Weak association constants between cytochrome C and 18-crown-6 in methanol (Ks = 1-3 M-1) have been reported by Odell and Earlam.[ref] These interactions did even result in solubilization of some proteins in polar organic solvents.
In this contribution we present an overview of the studies performed in our laboratories on the effects of crown ethers on the activity of enzymes in nonaqueous organic solvents. Three different types of enzymes, which have shown practical applications in organic solvents, viz. proteases, tyrosinase, and lipases, are discussed in this order. Different strategies of (pre)treatment with crown ethers in order to achieve enhancement of enzyme activity are dealt with in the section on proteases.
2. Materials
Crown ethers: 18-crown-6 (Shell), dibenzo-18-crown-6 (Aldrich), dicyclohexyl-18-crown-6 (Aldrich), monoaza-18-crown-6 (Aldrich), dibenzo-24-crown-8 (Fluka), diaza-18-crown-6 (Merck), 15-crown-5 (Merck), decyl-18-crown-6 (Merck), kryptofix 2.2.2 (Merck), and kryptofix 2.2 didecyl (Merck) were stored at 4oC and used as such. Pentaglyme was synthesized from pentaethylene glycol (Aldrich) by reaction with sodium hydride and methyliodide.
(-Chymotrypsin (E.C. 3.4.21.1), type II, from bovine pancreas (54 U/mg), subtilisin Carlsberg (E.C. 3.4.21.62), type VIII, from Bacillus licheniformis (10.4 U/mg), and trypsin (E.C. 3.4.21.4), type III, from bovine pancreas (10600 U/mg) were from Sigma. Cross-linked crystalline subtilisin Carlsberg was prepared according to the method of Schmitke et al.19 Tyrosinase (E.C. 1.14.18.1), from mushroom (24000 U/mg), and glass beads (150-212 micron) were purchased from Sigma. Lipases (E.C. 3.1.1.3) from Candida antarctica (3 U/mg), Aspergillus niger (1 U/mg), Candida cylindracea (32.3 U/mg), Mucor miehei (1.3 U/mg), Pseudomonas fluorescens (3414 U/mg), and Pseudomonas cepacia (48 U/mg) were obtained from Fluka.
The buffers used for the pretreatment of proteases, tyrosinase, and lipases are subsequently 20 mM Na2HPO4/NaH2PO4; pH 7.8, 100 mM KH2PO4/KOH; pH 7.0, and 20 mM Na2HPO4/NaH2PO4; pH 8.0. The enzyme preparations were stored at -20oC after pretreatment.
All solvents used are of analytical grade or higher and used as such.
N-Ac-L-Phe-OH, L-Phe-NH2, L-Tyr-NH2, and N-Ac-L-Phe-OEt were from Sigma. D-Phe-NH2 and L-Leu-NH2 were from Bachem. N-Ac-L-Phe-OEtCl was prepared from N-Ac-L-Phe-OH and 2-chloroethanol using Amberlite IR-120 as a catalyst. p-Cresol was obtained from Merck. Geraniol was from Aldrich and vinylacetate from Fluka.
3. Methods
This section describes a number of methods for treatment of different types of enzymes, viz. proteases, tyrosinase, and lipases, with crown ethers in order to enhance the enzymatic activity in organic solvents.
3.1 Proteases
Proteases have been intensively studied in water-poor organic solvents using transesterification reactions of N-protected amino acid esters.1 Also peptide bond formation reactions have been catalyzed by proteases in non-aqueous media, which are obviously of larger practical importance. In order to minimize hydrolysis both types of enzymatic reactions have to be performed under water-poor conditions. This has the drawback that the protease activity is generally low and therefore enhancement of the enzymatic activity under these circumstances is of large importance. Crown ether (pre)treatment of the enzymes has proven a very effective way for the enhancement of protease activity in organic solvents. Several ways for the pretreatment of normal protease preparations as well as cross-linked crystals of subtilisin Carlsberg will be described.
3.1.1 Crown ether addition to reaction solvent[ref],[ref]
As the structure of the crown ether determines to a large extent the complexation behavior (vide supra) it can be expected that different crown ethers have different effects on the enzyme activity. Table I shows the influence of the crown ether structure on the activity of the serine protease (a-chymotrypsin. In this study the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in toluene (0.006% H2O) was used as a model reaction (Scheme 1).INSERT SCHEME 1 The crown ethers were simply added to the substrate-containing reaction medium.¶ Table 1 clearly indicates that the 18-membered crown ethers are the most effective. Addition of 2 mM 18-crown-6 results in a 20 times higher enzyme activity. The 2 mM crown ether concentration was found to be optimal in case of 18-crown-6 with (a-chymotrypsin in toluene. The activation gradually increases until a plateau value for the activation of 20 times is reached at concentrations above 1.5 mM. The linear chain analog of 18-crown-6, pentaglyme (CH3-O-[-CH2-CH2-O-]5-CH3) has hardly any effect on the rate of the reaction. This shows that the acceleration of the enzyme-catalyzed reaction is mainly caused by a macrocyclic effect.
As shown in Table II most profound accelerations by crown ether are observed in the more hydrophobic solvents, such as octane, cyclohexane, dibutyl ether, and toluene. The intrinsic enzyme activity (in the absence of crown ether) is already higher in these solvents than in the hydrophilic solvents due to their lower ability to strip of the essential water layer from the enzyme.1 Although the crown ether activation effect is lower in hydrophilic solvents also in these solvents significant enhancements of enzyme activity can be obtained by the simple addition of 18-crown-6 to the reaction solvent.
INSERT TABLES I AND II
3.1.2 Pretreatment by lyophilisation in the presence of crown ethers[ref],[ref],[ref]
Biocatalysts that are used in organic media are generally lyophilized from an aqueous buffer, adjusted to the optimal pH value for the enzyme activity in water.[ref] The presence of several additives, such as the inhibitor N-Ac-L-Phe-NH2[ref], and the lyoprotectants polyethylene glycol13, sorbitol13, and ethyl cellulose[ref] during the lyophilisation process is advantageous to enhance protease performance in organic solvents. Activation factors up to 150 times have been reported.13We found that addition of 18-crown-6 prior to lyophilisation of (a-chymotrypsin has a significantly larger effect on the initial activity (Figure 1). In this method the protease (5mg/ml) is dissolved in a 20 mM Na2HPO4/NaH2PO4 buffer; pH 7.8 containing the indicated amount of crown ether. After incubation for 5 minutes the solution is quickly frozen in liquid nitrogen followed by lyophilisation for 24 hours. After separate equilibration of the enzyme powders, solvents, and substrates in a dessicator above a saturated salt solution at a thermodynamic water activity1 of 0.113 the initial rate of the enzyme reactions is measured. In this case the transesterification of N-Ac-L-Phe-OEt with 1-propanol (Scheme 1) in cyclohexane was chosen as a model reaction. The presence of 250 equivalents of crown ether with respect to the enzyme results in a 650 times enhanced enzymatic activity (Figure 1). As a result, the second order rate constant (kcat/Km) of the suspended enzyme towards N-Ac-L-Phe-OEt is 770 M-1.s-1. This is only 50 times lower than that of the (a-chymotrypsin-catalyzed hydrolysis reaction of this substrate in aqueous solutions. INSERT FIGURE 1
A practical problem was encountered in the lyophilisation studies of enzymes with the smaller crown ethers 12-crown-4 and 15-crown-5. Due to sublimation of these crown ethers during the lyophilisation process only a small fraction, 1 and 4% respectively, of the initial 500 equivalents of crown ether is retained in the resulting enzyme powder. Nevertheless, the resulting enzyme preparations showed a 20 and 50 time higher activity than those lyophilized in the absence of crown ether.
When the crown ether-pretreated enzyme powders are washed with non-aqueous solvents before application in organic media the activation is completely lost. Therefore, it can be concluded that the crown ether-induced activation is not a consequence of lyoprotection during the lyophilisation process.
The reaction rates described above (Tables I and II) are initial rates. From a practical point of view the product yields are more interesting. The effect of crown ether activation on product yields was investigated for the (a-chymotrypsin-catalyzed peptide bond formation between N-Ac-L-Phe-OEtCl and L-Phe-NH2 (Scheme 2). Figure 2 gives yields as a function of time of the dipeptide N-Ac-L-Phe-L-Phe-NH2 formation catalyzed by (a-chymotrypsin that was lyophilized in the absence and presence of 18-crown-6. After 8 h almost 70% of the dipeptide was formed with the crown ether-pretreated (a-chymotrypsin, whereas only 1.5% was formed in the case of the non-pretreated enzyme. Under initial conditions the crown ether-pretreated enzyme showed a 450 times higher activity, which corresponds to an enhancement from 1.5 * 10-3 U/mg to 0.7 U/mg of enzyme. Crown ether-pretreated enzyme can also be applied on a preparative (gram) scale. N-acetyl-L-phenylalanyl-L-phenylalaninamide could be isolated in 85% yield, from a reaction of N-acetyl-L-phenylalanine 2-chloroethylester and L-phenylalaninamide, using (a-chymotrypsin lyophilized in the presence of 50 equivalents of 18-crown-6 as catalyst, after 24 hours. 10INSERT SCHEME 1 AND FIGURE 2 The optimal amount of 18-crown-6 in the case of the peptide bond formation reactions, which are for solubility reasons performed in acetonitrile, is 50 molar equivalents with respect to the enzyme, whereas for the transesterification reactions in cyclohexane this amount is 500 equivalents (Figure 1). Most probably, the different hydrophobicities of the solvents are the reason for this difference. Since a distinct optimum in the amount of 18-crown-6 is observed in both cases, the determination of the optimal amount for each individual reaction and condition seems to be necessary.
The crown ether activation effect of protease-catalyzed peptide bond formation was found to be general. Also other serine proteases, such as subtilisin Carlsberg and trypsin, can be activated by lyophilisation in the presence of 18-crown-6. Furthermore, the data for the dipeptides in Table III show that in all cases the rate of the (a-chymotrypsin-catalyzed peptide bond formation is enhanced with factors ranging from approximately 200 in the case of the 2-chloroethyl ester of N-acetyl-L-phenylalanine with L- and D-phenylalanamide to 100 with L-tyrosinamide as the nucleophile.
INSERT TABLE III
3.1.3 Pretreatment of enzymes by acetone precipitation in the presence of crown ethers
Precipitation of proteins from aqueous buffers by trituration with cold acetone is a well-known technique in protein chemistry. After precipitation, followed by centrifugation and decanting of the supernatant, the resulting enzyme powder is washed several times with cold acetone. After solvent evaporation the dried enzyme powder is obtained. This way of drying proteins is relatively fast compared to lyophilisation.
The crown ether can be added at three different stages during this precipitation procedure, (i) addition to the aqueous buffer, (ii) together with the acetone added for the precipitation, or (iii) together with the washing acetone. All possible combinations were investigated and the results are given in Table IV. After equilibration of solvent, substrates, and corresponding enzyme powders at aw = 0.113 the enzymatic activities were determined for the peptide bond formation reaction with N-Ac-L-Phe-OEtCl and L-Phe-NH2 in acetonitrile (Scheme 2). The specific activity of the (a-chymotrypsine sample which is obtained from the acetone precipitation procedure without any addition of crown ether is 4.1 nmol/min*mg of enzyme. This is almost three times higher than the activity of lyophilized (a-chymotrypsin powder and shows that acetone precipitation is a fast and useful alternative for pretreatment of enzymes for applications in organic media. A further tenfold increase in activity is observed when 18-crown-6 is present during the washing procedure, despite the fact whether the 18-crown-6 has been present or absent during the actual precipitation. On the other hand, a tenfold decrease in activity is found in cases where (a-chymotrypsin was precipitated in the presence of 18-crown-6 and subsequently washed in the absence of 18-crown-6. This is in accordance with the observations with the crown ether-activated lyophilized a-chymotrypsin powders which also lost most of their activity upon washing with organic solvent (vide supra). Crown ether activation of a-chymotrypsin by acetone precipitation can therefore only be achieved if the crown ether is present in the final, dry enzyme powder.
INSERT TABLE IV
3.1.4 Pretreatment of cross-linked crystalline subtilisin Carlsberg[ref]
A new and promising technology to improve the operational stability of enzymes in organic solvents is the use of cross-linked enzyme crystals.[ref] Although also such crystals usually have a relatively low catalytic activity in organic media, they are highly resistant against autolysis and thermal inactivation. Cross-linked enzyme crystals therefore can be reused in many reaction cycles without appreciable loss of activity.
The effect of 18-crown-6 on the activity of cross-linked enzyme crystals of the serine protease subtilisin Carlsberg[ref],[ref] was investigated by three methods: (i) simple addition of the crown ether to the enzymatic reaction medium, (ii) pretreatment by lyophilisation of the protein crystals in the presence of crown ether, and (iii) soaking and drying of the crystals from a crown ether solution in acetonitrile. The peptide bond formation reaction between N-Ac-L-Phe-OEtCl and L-Phe-NH2 in acetonitrile (Scheme 2) was used as the model reaction. Simple addition of 18-crown-6 to the reaction medium appeared to have no effect on the rate of the enzyme-catalyzed peptide bond formation (Table V). Lyophilisation of the enzyme crystals in the presence of 18-crown-6, however, showed some interesting effects (Table V). Whereas lyophilisation in the absence of crown ether results in a decrease to less than 10 % of the original enzyme activity, the presence of 50 mmol of 18-crown-6 during the lyophilisation process yields a sample with a 8.5 times larger activity. The reduction of the activity after lyophilisation of the enzyme crystals might originate from a distortion of the enzyme conformation by crystallization of the water in the solvent-filled channels of the crystals. The presence of 18-crown-6 might prevent this process and in this respect 18-crown-6 may act as a lyoprotecting agent. However, the enhancement by a factor of 8.5 indicates that 18-crown-6 must have an additional role as an activator of the enzyme catalysis.
INSERT TABLE V
The third way of enzyme pretreatment that was investigated was soaking of the cross-linked enzyme crystals in a crown ether solution of acetonitrile, followed by gentle evaporation of the solvent. The enzymatic activities given in Table VI for the rates of peptide bond formation of N-Ac-L-Phe-L-Phe-NH2 show that subtilisin Carlsberg crystals can be significantly activated by this procedure. The initial activity of the crown ether-activated enzyme crystals is almost equal in the case of lyophilisation (Table V, entry 6) and the soaking and drying strategy (Table VI, entry 2). This suggests that the same molecular mechanism is responsible for the observed activation in these cases.
Also in this case the most effective crown ethers are 18-crown-6 and monoaza-18-crown-6, with rate enhancements of about ten. Treatment with more hydrophobic and sterically hindered crown ethers, like dibenzo- and decyl-18-crown-6, is less effective. The macrocyclic effect is also manifest for cross-linked crystalline enzymes as pretreatment with pentaglyme gives only a twofold rate enhancement.
The crown ether activation of the cross-linked enzyme crystals was found to be very persistent in time. No loss of activity was found for crown ether-activated cross-linked crystalline subtilisin Carlsberg, even after storage for four weeks at 4°C.
INSERT TABLE VI
Figure 3 shows the influence of the crown ether concentration present in the soaking and drying procedure on the activation of cross-linked subtiline Carlsberg crystals. At a concentration of 50 mM 18-crown-6 a plateau is reached at which the enzyme activity is enhanced 13 times. This concentration corresponds to about 1350 mol equivalents of 18-crown-6 with respect to the enzyme.
INSERT FIGURE 3
3.2 Tyrosinase[ref]
The advantages of enzymatic catalysis in organic solvents were clearly shown in the tyrosinase-catalyzed oxidation of p-cresol to the corresponding o-quinone (scheme 3).[ref] This reaction cannot be performed in aqueous media since o-quinones polymerize spontaneously under these conditions. Moreover, this polymerization reaction inactivates the enzyme.
INSERT SCHEME 3
Since the lyophilized enzyme powder tends to stick to the glass wall of the reaction vessel, tyrosinase was immobilized on glass beads prior to equilibration at aw = 0.33.21 The effect of several macrocycles on the enzymatic activity of tyrosinase is given in Table VII. Macrocycles were directly added to the reaction medium, chloroform and tetrachloromethane, and in both media this results in a considerable enhancement of the tyrosinase activity, up to 70 times. In addition, no effect was observed upon addition of pentaglyme, the open chain analog of 18-crown-6, again demonstrating the macrocyclic nature of the activation. This shows that macrocyclic activation is also applicable for immobilized enzymes.
Kryptofix 2.2.2 is found to be the most effective macrocycle in both solvents and the other macrocycles investigated have about equal effects in chloroform. In tetrachloromethane monoaza-18-crown-6 is significantly less effective than the other macrocycles. On the other hand, in hexylacetate, a solvent in which tyrosinase has a tenfold higher activity than in chloroform, no macrocycle activation is found. This shows, as in the case of proteases (vide supra), that for each enzyme, type of reaction, and solvent, the conditions should be carefully selected in order to obtain the maximal macrocycle activition.
INSERT TABLE VII
Crucial factors for crown ether activation are not only the solvent, enzyme, and type of reaction, but also the thermodynamic water activity. Table VIII gives the effects of macrocycles in chloroform at a thermodynamic water activity of 0.72. In this case no activation by addition of macrocycle is found, while the activation is significant at aw = 0.33 (Table VII). However, it should be noted that the initial tyrosinase activity in the absence of macrocycle is already 400 times higher in the case of aw = 0.72. In general, a higher water activity will enhance the enzyme flexibility of the enzymes in organic solvents which leads to enhanced enzymatic activity.[ref] However, these conditions are not applicable in the tyrosinase-catalyzed oxidation since the presence of water in the reaction medium leads to significant formation of byproducts as observed by UV-spectroscopy.
INSERT TABLE VIII
3.3 Lipases
The use of lipases in nonaqueous media is one of the most promising methods for the enantioselective conversion of a wide variety of hydrophobic acids and alcohols.[ref] Lipases generally have a broad substrate specificity while maintaining a high enantioselectivity. Most of them posses an active site-covering lid and require interfacial activation, i.e. opening of the lid at a water-lipid interface.
Lipases are ubiquitous enzymes and can be of mammalian, fungal, and bacterial origin. The effect of 18-crown-6 on lipase activity has been investigated for different lipases (Table IX). In this case 18-crown-6 was added prior to lyophilisation of the enzymes. The acetylation of geraniol with vinylacetate in hexane[ref] was used as a test reaction (Scheme 4). The solvent and the pretreated enzyme preparations were equilibrated at aw = 0.113. The results show that crown ether-induced activation of the enzymatic activity is only significant in cases where the (commercial) lipase preparations are generally of high specific activity, e.g. Candida cylindracea, and Pseudomonas fluorescens lipase. Only minor activation or sometimes even a deactivation is observed with less pure lipase samples. The low specific activity lipase preparations (<10 U/mg) generally contain only a few percent of protein material and the absence of a crown ether effect is probably due to complexation of 18-crown-6 with the bulk material, which mainly originates from biological membranes. After the observation that the purity of the lipase samples is very important for the crown ether activation additional experiments were perfomed with high purity Pseudomonas fluorescens lipase (3414 U/mg).
INSERT TABLE IX
Figures 4a-c show the activity of Pseudomonas fluorescens lipase lyophilized with different amounts of 18-crown-6 in acetonitrile (aw = 0.113), acetonitrile (aw = 0.753), and hexane (aw = 0.113). Although the activity of the enzyme lyophilized in the absence of crown ether is different due to differences in hydrophobicity and thermodynamic water activity of the media, the activation factor is almost identical in the three cases. This suggests that the same activation mechanism is operative under these conditions. Like in the case of the proteases, also here the enzyme activation was completely lost after washing of the crown ether-activated enzyme powder with dry ethyl acetate.
INSERT FIGURE 4A, B, AND C
3.4 Possible origin of the crown ether activation
The mechanism of the crown ether activation on enzyme suspensions in organic solvents is not fully understood. However, we have proposed two effects which may contribute to the enhancement of the enzyme activity by crown ethers.8,10,16 Firstly, crown ethers can form complexes with quarternary ammonium groups4 of lysine residues of the enzyme, thereby preventing the formation of salt-bridges of these groups with oppositely charged amino acid residues in the enzyme powder. In organic solvents the formation of such inter- and intramolecular salt bridges is relatively strong and may either destabilize the enzyme[ref] or lock it in a catalytically inactive conformation. Conformational changes are necessary for intramolecular salt bridge formation. Since enzymes display a large conformational rigidity in organic solvents intramolecular salt bridge formation seems to be less important in these media. Intermolecular salt bridge formation may lead to unfavorable protein-protein interactions, which prevent the accessibility of the active site. Therefore, inhibition of salt-bridge formation by complexation with crown ether molecules may result in a more active enzyme.
Secondly, crown ethers may contribute to an enhanced substrate binding and consequently to a higher enzymatic activity by facilitating the transport of water molecules from the active site into the bulk organic solvent. Binding of the substrate to the active site requires (partial) dehydration.[ref] In aqueous solutions the transfer of water molecules from the active site to the bulk solvent is entropically favorable due to an increase in translational and rotational freedom.[ref] However, in organic solvents, especially when these have a low polarity, dehydration of the active site is an overall unfavorable process. Accompanied by an altered partitioning of the substrate and product molecules between the bulk organic phase and enzyme active site[ref],[ref] this will result in a much weaker binding of the substrate by the enzyme. Increased Km values and thus a lowered enzymatic activity are found upon transfer of enzymes from an aqueous solution into an organic solvent.[ref] Crown ethers, that are able to complex water molecules in organic solvents, will facilitate the transport of water from the active site to the bulk organic solvent during the reaction. Golovkova and coworkers have determined the complexation ability of several crown ethers with water in CDCl3.[ref] Complexation decreased in the order dicyclohexyl-18-crown-6 > 18-crown-6 > dibenzo-18-crown-6 > dibenzo-24-crown-8 > 15-crown-5. This is in line with the effects of crown ethers on the enzyme activity (Table I and IV), taking into account the differences in partitioning which are related to their hydrophobicity and steric demands.
4. Concluding remarks
Enzymatic catalysis in nonaqueous solvents is a useful tool for organic synthesis, especially for reactions in which optical resolution steps are involved. Due to the intrinsically low catalytic activity of enzymes in these environments, generally two to six orders of magnitude lower than in water, practical methods for enzyme activation are of high importance. We have shown that the (pre)treatment of enzymes with crown ethers is a simple and effective way to increase enzymatic activity in organic media up to a level which is only one to two orders of magnitude lower compared to their activity in water. Crown ether induced activation is shown to be generally applicable for various enzymes, and different types of enzyme preparations, with a wide variety of solvents, reaction conditions, and reactions. Therefore this method gives good prospects for practical applications, as the major problem of enzymatic catalysis in non-aqueous media, i.e. a too low enzymatic activity, can be overcome very effectively by this methodology.
5. Acknowledgement
The Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NOW) and the Technology Foundation STW are acknowledged for financial support.
¶ As most crown ethers are quite hygroscopic and are stored at 4oC, the crown ether containing vessels should be kept closed during equilibration to ambient temperature.
1 Halling, P.J. (1992) Salt hydrates for water activity control with biocatalysts in organic media. Biotechnol. Techniques, 6, 271-276.
??18
Table I Influence of various crown ethers on the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol, catalyzed by a-chymotrypsine in toluene.a
Crown ether Activation
(V0(crown
ether)/V0(blank)b
18-Crown-6 19.6
Monoaza-18-crown-6 11.3
Dibenzo-18-crown-6 6.0
Dicyclohexyl-18-cro 4.0
wn-6
2.2 Didecyl 2.6
Kryptofix
Decyl-18-crown-6 1.9
Dibenzo-24-crown-8 1.8
15-Crown-5 1.8
Pentaglyme 1.4
aa-Chymotrypsin (5 mg/ml) was lyophilized from 100 mM K2HPO4/KH2PO4 buffer; pH 7.8. Model reaction: 2.5 mM ester, 1 M 1-propanol, 2 mM crown ether, 0.5 mg/ml pretreated enzyme, 25oC. b Vo(blank) = 0.50 mM.min-1.
Table II Influence of 18-crown-6 on the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol, catalyzed by a-chymotrypsine in various solvents.a
Solvent V0(blank) V0(2 mM Activation
(mM.min-1) 18-C-6)
(mM.min-1)
Octane 5.4 ± 0.6 154.5 ± 29
8.0
Cyclohexane 4.2 ± 0.8 80.5 ± 9.5 19
Dibutyl 0.17 ± 5.3 ± 0.4 31
ether 0.01
Toluene 0.50 ± 9.8 ± 1.3 20
0.02
t-Amylalcohol 0.02 ± 0.04 ± 2
0.00 0.00
THF + 1% H2O 0.18 ± 1.45 ± 8
0.00 0.05
a a-Chymotrypsin (5 mg/ml) was lyophilized from 100 mM K2HPO4/KH2PO4 buffer; pH 7.8. Model reaction: 2.5 mM ester, 1 M 1-propanol, 2 mM 18-crown-6, 0.5 mg/ml pretreated enzyme, 25oC.
Table III Initial rates of peptide bond formation by a-chymotrypsin, lyophilized in the presence or absence of 18-crown-6.a
Dipeptide Vob Vob Activation
(No (50 Equivalents
18-crown-6) 18-crown-6)
N-Ac-L-Phe-L-Phe-NH2 0.22 39.6 181
N-Ac-L-Phe-D-Phe-NH2 0.29 58.6 204
N-Ac-L-Phe-L-Leu-NH2 0.54 72.5 135
N-Ac-L-Phe-L-Tyr-NH2 0.62 65.7 106
aa-Chymotrypsin (5 mg/ml) was lyophilized from 20 mM Na2HPO4/NaH2PO4 buffer; pH 7.8 in the presence of the indicated amount of 18-crown-6. Model reaction: 5 mM of N-Ac-L-Phe-OEtCl and amino acid amides, 1 mg/ml pretreated a-chymotrypsin, acetonitrile, aw = 0.113, 30°C. b V0 are in nmol/min/mg of enzyme.
Table IV Influence of 18-crown-6 addition at different stages during acetone precipitation of a-chymotrypsin.a
Crown ether addition V0 V0/V0(bla
(nmol/min*mg nk)
of enzyme)
Buffer Precipitat Washing
ion acetone
acetone
- - - 4.1 1
+ - - 0.3 0.1
- + - 0.7 0.2
- - + 40.3 10
+ + - 0.5 0.1
+ - + 33.2 8
- + + 36.6 9
+ + + 35.7 9
a Precipitation: 10 mg a-chymotrypsin in 1 ml 20 mM Na2HPO4/NaH2PO4 buffer; pH 7.8 was precipitated with 2 ml cold acetone. The resulting enzyme powder was washed 3 times with 1 ml acetone, followed by solvent evaporation. If 18-crown-6 was present during the different stages in the process the concentration was 40mM. Model reaction: 50 mM N-Ac-L-Phe-OEtCl and L-Phe-NH2, 2.5 mg/ml enzyme powder, acetonitrile, aw = 0.113, 30oC.
Table V Effects of 18-crown-6 on cross-linked crystalline subtilisin Carlsberg by crown ether addition to the reaction mixture or by pretreatment with lyophilisation.a
Pretreatment 18-crown-6 V0
(mM) (nmol/min*mg)
Addition to the 0 0.45
reaction medium 12.5 0.50
50.0 0.43
Lyophilisation 0 0.03
12.5 1.07
50.0 3.80
a Lyophilisation: 2.5 mg subtilisin Carlsberg crystals were lyophilized from 500 ml 20 mM Na2HPO4/NaH2PO4 buffer; pH 7.8 containing the indicated amount of 18-crown-6. Model reaction: 50 mM N-Ac-L-Phe-OEtCl and L-Phe-NH2, 2.5 mg/ml enzyme crystals, acetonitrile, aw = 0.113, 30oC.
Table VI Effects of soaking and drying of cross-linked crystalline subtilisin Carlsberg from a solution of crown ether in acetonitrile on the rate of peptide bond formation.a
Crown ether V0 Activation
(nmol/min*mg)
None 0.43 -
18-Crown-6 4.11 9.6
Monoaza-18-crown-6 4.82 11.2
15-Crown-5 1.25 2.9
Decyl-18-crown-6 0.54 1.2
Dicyclohexyl-18-crown-6 1.64 3.8
Dibenzo-18-crown-6 0.81 1.9
Dibenzo-24-crown-8 0.38 0.9
Pentaglyme 1.04 2.4
a Enzyme crystals (1 mg/ml) were dried from acetonitrile containing 15 mM of crown ether. Model reaction: 50 mM N-Ac-L-Phe-OEtCl and L-Phe-NH2, 0.5 mg/ml enzyme crystals, acetonitrile, aw = 0.113, 30oC.
Table VII Influence of various macrocycles on the initial rate of the tyrosinase-catalyzed oxidation of p-cresol in halogenated solvents at aw = 0.33.a
Macrocycle V0(macrocycle)/V0
Chloroformb Tetrachlorometha
nec
Kryptofix 2.2.2 59.4 ± 0.9 70.9 ± 3.0
18-Crown-6 21.0 ± 2.0 58.6 ± 6.0
Monoaza-18-crown-6 18.9 ± 0.1 4.4 ± 0.7
Dicyclohexyl-18-cr 17.0 ± 2.0 19.1 ± 0.0
own-6
aConditions: 20 mM p-cresol, 50 mg/ml immobilized tyrosinase, 3 mM macrocycle, aw = 0.33, 25oC.
b V0 = 1.45*10-7 M min-1, c V0 = 1.13*10-8 M min-1
Table VIII Influence of various macrocycles on the initial rate of the tyrosinase-catalyzed oxidation of p-cresol in chloroform at aw = 0.72.a
Macrocycle V0(macrocycle)
/V0a
Kryptofix 2.2.2 1.02 ± 0.09
18-Crown-6 1.20 ± 0.09
Monoaza-18-crown-6 1.51 ± 0.04
aConditions: 20 mM p-cresol, 50 mg/ml immobilized tyrosinase, 3 mM macrocycle, aw = 0.72, 25oC.
b V0 = 6.14*10-5 M min-1
Tabel IX Influence of lyophilisation of various lipases in the presence of 18-crown-6 on the enzymatic activity in the acetylation of geraniol.a
Lipase Conc. Activity
18-crown-6 (nmol/min*mg)
(mM)
Candida antarctica 0 51.4 ± 1.8
(3U/mg) 5 61.4 ± 2.6
25 82.8 ± 4.8
Aspergillus niger 0 185 ± 11
(1U/mg) 5 196 ± 7
25 34 ± 3
Candida cylindracea 0 6.6 ± 1.8
(32.3U/mg) 5 17.7 ± 2.5
25 33.6 ± 3.9
Mucor miehei 0 0.15 ± 0.02
(1.3U/mg) 5 0.18 ± 0.01
25 0.05 ± 0.01
Pseudomonas 0 1305 ± 94
fluorescens
(3414U/mg) 5 4713 ± 370
25 5530 ± 485
Pseudomonas cepacia 0 309 ± 58
(48U/mg) 5 550 ± 58
25 228 ± 76
aConditions: 2 mg/ml lipase in 20 mM Na2HPO4/NaH2PO4-buffer; pH 8.0 was lyophilized in the presence of the indicated amounts of 18-crown-6. Reaction conditions: 0.4 mg/ml enzyme, 50 ml geraniol,
92 ml vinylacetate, 5 ml hexane; aw = 0.113; 30oC.
Figure 1 Crown ether activation (V0(18-crown-6)/V0) of transesterification catalyzed by -chymotrypsin as a function of the amount of 18-crown-6 present during lyophilization. -Chymotrypsin (5 mg/ml) was lyophilized from 20 mM Na2HPO4/NaH2PO4 buffer; pH 7.8 in the presence of the indicated amount of 18-crown-6. Reaction conditions: 2.5 mM N-Ac-L-Phe-OEt, 1 M 1-PrOH, cyclohexane, aw = 0.113, 25oC.
Figure 2 Enzymatic synthesis of N-Ac-L-Phe-L-Phe-NH2 catalyzed by -chymotrypsin in acetonitrile. Circles: enzyme lyophilized in the presence of 50 molar equivalents of 18-crown-6; triangles: enzyme lyophilized without crown ether. -Chymotrypsin (5 mg/ml) was lyophilized from 20 mM Na2HPO4/NaH2PO4 buffer; pH 7.8. Reaction conditions: 50 mM N-Ac-L-Phe-OEtCl and L-Phe-NH2, acetonitrile, aw = 0.113, 30oC.
Figure 3 Crown ether activation (V0(18-crown-6)/V0) for the peptide bond formation catalyzed by cross-linked subtilisin Carlsberg crystals in acetonitrile as a function of the amount of 18-crown-6 present during soaking and drying of the enzyme crystals. Subtilisin Carlsberg crystals were (1 mg/ml) were soaked and dried from acetonitrile containing the indicated concentration of 18-crown-6. Reaction conditions: 50 mM N-Ac-L-Phe-OEtCl and L-Phe-NH2, acetonitrile, aw = 0.113, 30oC.
Figure 4 Effect of 18-crown-6 on the Pseudomonas fluorescens lipase catalyzed acetylation of geraniol as a function of the presence of 18-crown-6 during lyophilisation of the enzyme and reaction conditions. Curve A represents acetonitrile at aw = 0.113, curve B acetonitrile at aw = 0.753, and curve C hexane at aw = 0.113. Pseudomonas fluorescens lipase (1 mg/ml) was lyophilized from 20 mM Na2HPO4/NaH2PO4 buffer; pH 8.0 in the presence of the indicated concentration of 18-crown-6. Reaction conditions: 50 ml geraniol, 92 ml vinylacetate, 5 ml solvent at the indicated thermodynamic water activity, 30oC.
Scheme 1 Protease-catalyzed transesterification of the ethyl ester of N-acetyl-L-phenylalanine with 1-propanol.
Scheme 2 Protease-catalyzed peptide bond formation between the 2-chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide.
Scheme 3 Tyrosinase catalyzed oxidation of p-cresol to o-quinone.
Scheme 4 Lipase-catalyzed acetylation of geraniol.