BIOTECHNOLOGY LETTERS Volume 15 No.12 (Dec.1 993) pp.1223-1228 Received as revised 3rd November

SYNTHESIS OF GTYROSINE GLYCERYL ESTER CATALYZED BY dXYMOTRYPSIN IN WATER-MISCIBLE ORGANIC SOLVENTS:

A POSSIBLE SUN-TAN ACCELEitATOR PRODUCT

Pedro Lozano, Didier Combes +, Jose L. Iborra* and Arturo Manj6n

Departamento de Bioqufmica y Biologfa Molecular B e Inmunologia. Facultad de Quimica. Universidad de Murcia. Ap. 4021. E-30001. MURCIA. Esparia.

+DCpartement de Genie Biochimique et Alimentaire. INSA UA-CNRS 544. Complexe Scientifique de Rangueil. F-3 1077. TOULOUSE. France.

SUMMARY The synthesis of L-tyrosine glyceryl ester, from glycerol and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by a-chymotrypsin. Values of 60 % (v/v) for glycerol and 200 mM for L-tyrosine methyl ester were optimal for the transesterification reaction. Additionally to glycerol, several other water miscible cosolvents (acetonitrile, N,N’-dime@ formamide and tetrahydrofurane) were tested in the reaction media, but their presence did not give an enhancement on the transesterification activity with respect to the glycerol/water medium. However, increasing the hydrophobic@ of the cosolvent resulted in a reduction of the enzyme activity, the water:glycerol mixture being the best reaction media.

INTRODUCTION

Peptides and amino acid derivatives have important applications in food and pharmacological areas. In the cosmetic industry, hydrophilic L-tyrosine derivatives have a great interest because of their properties as sun-tan accelerators. The very poor solubility of L-tyrosine in water limits its direct use for cosmetic formulations. Thus, recently, the use of hydrophilic L- tyrosine derivatives synthesized by enzymic ways as sun-tan accelerators in novel cosmetic formulations has been described (Monsan and Paul, 1990). In fact, L-tyrosine is the first substrate to produce the skin pigmentation, which is firstly hydroxylated to L-DOPA, and then oxidized to L-dopaquinone (the key intermediate in the melanin pigmentation) by the consecutive action of both the hydroxylase and oxidase activities of tyrosinase. The use of proteases to catalyze peptide bonds or amino acid esters synthesis in hydrophilic organic solvents has been extensively reported (Ingalls et al., 1975; Mori et a,!, 1987; Rise et al, 1990, Cardillo-Theobaldo et ui, 1991; Lozano et aL, 1992). There are many advantages in employing one-phase liquid cosolvent systems for enzymic synthesis, such as high reactants

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solubilities, shift of reaction equilibria by controlling water activity, and absence of diffusional limitations across water/organic solvent interfaces (Phillips er al., 1990, Kise and Hayakawa, 1991; Lozano and Combes, 1992). The aim of this paper was to study the synthesis of an hydrophilic L-tyrosine derivative, as the L-tyrosine glyceryl ester, by a transesterification reaction catalyzed by cr-chymotrypsin. The influence of the substrate concentration, the glycerol content, as well as the presence of other water-miscible cosolvents, was analyzed.

MATERlALS AND METHODS Materials cr-Chymotrypsin (EC 3.4.21.1., type II from porcine pancreas) and L-tyrosine methyl ester were obtained from Sigma Chem. Co., and used without previous purification. Glycerol, N.N’- dimethyl formamide, tetrahydrofurane and acetonitrile were Merck, analytical grade. Hydrolysis/transesteritication reactions Into an Eppendorf tube of l-ml total volume, 200 ~1 of 1 M L-tyrosine methyl ester dissolved in 0.2 M phosphate buffer pH 7.0, was placed. The reaction volume was adjusted to 980 ~1 by addition of the corresponding volume of water, glycerol or organic cosolvent, and then, 20 ~1 of a 2.5 mg/ml cw-chymotrypsin solution in water added. The reaction mixture was incubated without stirring at 40 Oc. Aliquots of 50 ~1 were extracted at several time intervals from the reaction mixture, previously homogenized by mechanical shaking, and mixed with 950 ~1 of 10 % (w/v) trichloroacetic acid (TCA) to stop the reaction. TCA-treated samples were immediately centrifuged (10 min at 2,800 g) at 6 OC to separate the precipitated protein and stored at -20 OC until analysis. HPLC analysis Substrates and products concentrations were determined by HPLC. An AminoQuant (model 1090, Hewlett-Packard) chromatograph, equipped with a Nucleosyl C-18 column (Touzan and Matignon, 25 cm length and 3.9 mm internal diameter, 5 pm particle size, and 10 nm pore size), was used. Samples were eluted isocratically with water:acetonitrile:acetic acid (89:lO: 1, v/v/v) at 1 ml/min flow rate and elution profiles detected at 280 nm. One unit of activity was defined as the amount of enzyme which produced 1 pmol of the hydrolysis (L-tyrosine) or transesterification (L-tyrosine glyceryl ester) product per minute

RESULTS AND DISCUSSION Synthesis of Gtyrosine glyceryl ester. As a kinetically-controlled process, the degree of hydrolysis of an amino acid ester by a serine protease is highly dependent on the water (nucleophile acceptor of the aminoacyl-enzyme complex) content of the reaction medium Thus, the presence of water-miscible organic solvents, able to reduce the water content/activity, should affect negatively the hydrolytic action of the enzyme, and favour the transfer of the acyl moiety of the acyl-enzyme intermediate complex to another nucleophile in the reaction medium. Thus, the influence of

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glycerol both a nucJcophile and a water-miscible organic solvent, on the a-chymouypsin activities on Ltyrosine methyl ester was studied.

0 5 10 13

Tlmo ( min )

FIgam 1. HFIX Chromalogram of the enzymatic synthesis of Ltyrosine giycmyl ester (TGE) from Ltyrosinc methyl ester (‘ME). (T): Lryrusine.

Tima ( mln )

Flgun 1. Reaction protilt of the u-chymotrypsin a&on on L- tyrosine methyl ester ( l ) to produce ttyrwiae ( A ) and L- fpsine glycql ester (8), using 100 mM ttyrosine methyl ester and 60 % (v/v) giyccrol in 0.2 M pbsphatc buFer pH 7.0

Figure 1 presents the HPLC chromatogram of one aliquot of a glycerol-containing reaction mixture, showing the appearance of three fraction clearly separated: (i) the hydrolytic product of the reaction (Ltyrosine), (ii) a new fraction attributed to the transesterifkatioa product (Ltyrosine glyceryl ester), and (iii) the acyl substrate of the reaction (L-tyrosine methyl ester). The overall mass balance of the reaction media during the full reaction time and the isolated products allowed to establish the corresponding standard calibration curve of the transesterification product with a correlation degree of 0.995. Figure 2 shows the reaction time-course for a typical experiment for the Ltyrosine gIycery1 ester synthesis catalysed by PI- chymotrypsi~~ in a medium containing 85 % (v/v) glycerol. As it can be seen, after 75 minutes of reaction time, the ester substrate was fully consumed. The synthesis of this Ltyrosine glycery1 ester by a-chymotrypsin in a medium containing glycerol was previously described by Ingalls er al (1975), as a contamination product arising during the esterification reaction of L tyrosine with ethanol, when glycerol was used as a water content reducing cosolvent. E&C% of water/glycerol content and substrate concentration.

The water content of ?be reaction medium in an enzymic transesterifkation process is an important factor since it will influence both the enzyme action and the equilibrium between substrate and product (Kise et ol, 1990). At low water content, the enzyme is expected to function less efficiently. However, a low water content should favour the shift of the equiiibrium towards synthesis (Luzano and Combes, 1992).

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Figure 3 shows both transesterification and hydrolytic activity profiles of cr-chymotrypsin as a function of the glycerol concentration. As it can be seen, the hydrolytic activity decreased proportionally to the increase in glycerol concentration. Additionally. the transesterification activity profile showed a maximum of enzyme activity at a 60 % (v/v) glycerol concentration. Thus, since tbe glycerol content of the reaction media had simultaneously a double role, as water content reducing agent and as a nucleophile acceptor of the aql enzyme intermediate, its presence had an additive effect on the enhancement of the transesterification activity. The decrease on the enzyme activity at glycerol concentrations higher than 60 % (v/v) could be attributed to the observed rise of the viscosity on the reaction media, resulting in an increase on the diffusional limitations and a less efficient enzyme action.

[Glycerol] ( # )

Figure 3. Effect of glycerol concentration on both the transesterification ( l ) and hydrolytic ( A ) activities of a-chymobypsin using L4yrosinc

methyl ester (200 mhI) as substrate.

n 3 -soa

.- i g 200 a

P E loo e I- n

5 0

Flgure 4. Effect of Ltyrosine methyl ester concentration on both the transesterihtion (0) and hydrolytic (A ) achities of a-chymotrypsin in 60 Q (v/v) glycerol.

The influence of the Ltyrosine methyl ester concentration on both the transesterificatioo and the hydrolytic activities of a-chy-motrypsin at 60% (v/v) glycerol concentration was also studied (Figure 4). Michaelis-Menten type kinetics was observed and thus the kinetic parameters could be derived from Hanes-Woolf plots, which values are summarized in Table I. As it can be seen in Figure 4, optimal conditions for the transesterifkation reaction were attained above 150 mM Ltyrosine methyl ester and 60% (v/v) glycerol, being the maximal rate 26.5times higher than the hydrolytic rate. Since the acylation of the enzyme by the amino acid ester substrate is identical in both the transesterifkation and the hydrolytic processes, the large difference in V,, should be attributed to the decrease in water content and water

activity in the reaction media produced by the presence of glycerol (Combes and Lozano, 1992). Additionally, it is noteworthy that the value of I$, for transesterification is about 2.3-

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times larger than for hydrolysis. Assuming that the binding of the ester substrate to the enzyme is equal in both processes, this K, difference could be attributed to the increase in

the diffusional limitations caused by the high viscosity of the reaction media.

Table 1. Kinetic parameters for both the transesterihtion and hydrolytic reactions catalyzed by a-chymotrypsin using L-tyrosine methyl ester as substrate in preseoce of 60 535 (v/v) glycerol in the reaction media.

ACTMTY “mar KM

@mol.min%g~l) (mM)

HIDROLYSIS r 123 23.6

TRANSESTERlFICATION 326.1 55.6

Thus, by means of a careful control of the medium conditions with respect to substrate concentration and glycerol content, it was possible to synthesize L-tyrosine glyceryl ester with a transesterification product yield of 80 %, and at a rate higher than for the normal hydrolytic activity of the enzyme. Influence of water miscible organic cosolvents In order to discriminate the double role of glycerol on the L-tyrosine glyceryl ester synthesis catalyzed by cr-chymotrypsin, the influence of other water-miscible organic solvents (acetonitrile, tetrahydrofurane and N,N’-dimethyi formamide) was also studied. Starting from a medium having the optimal conditions for transestetification, that is 60 % (v/v) glycerol, 40 % (v/v) water content, and 200 mM L-tyrosine methyl ester, the influence of these cosolvents has been studied by decreasing the glycerol concentration and keeping constant the water content into the reaction media, and thus obtaining one-liquid-phase systems with different glycerol/cosolvents [60 % (v/v) total content] mixtures. Figure 5 shows the transesterification activity profile of the a-chymotrypsin as a function of the solvents composition of the reaction media. As it can be seen, in all cases, the increase in glycerol concentration produced an enhancement of the transesterification activity, evidence of the role of glycerol as substrate. Additionally, at the same glycerol concentration, the presence of other cosolvents produced a decrease in the enzyme activity in such a way that an increase in the hydrophobic@ of the cosolvent was followed by a reduction on the transestetification activity of the enzyme, the transesterification activity following the order: water > NJ’-dimethyl formamide > acetonitrile > tetrahydrofurane. These results are in agreement to other authors (Phillips et al, 1990; Kise et al, 1990), where the increase in the hydrophobic@ of the reaction media resulted in a decrease in the different synthetic actions of cr-chymotrypsin (peptide synthesis, esterification and transesterification reactions).

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[Corohmnt] ( W )

F@re 5. Infhmee of several water-miscible cosoknts [(I ) water, ( A ) N,N’-dimcthyI formamide. (a) acctonitrilc, and (0) tctrahydrofuranc] on the transestcrihtion activity of a-ehymotrypsin using 200 mhl Llyrosine methyl ester as substrate.

CONCLUSIONS. a-Cbymotrypsin was able to catalyze tbe synthesis of Ltyrosine glyceryl ester by a transesterification process in a.reaction media containing glycerol. The maximal activity level was obtained for 200 mM Ltyrosine methyl ester, 60 % (v/v) glycerol and 40 % (v/v) water content. In such a medium, the transesterification products were fully soluble, resulting in a

clear reaction media which product concentration was largely higher than the solubility of L tyrosine in water. As it is known, glycerol is an important component in some cosmetic formulations, thus, the transesterification reaction mixture could be used for such preparations, and taking advantage of the sun-tan properties of Ltyrosine glyceryl ester.

ACKNOWLEDGEMENTS. P. Lozano is a postdoctoral reincorporation fellow of M.E.C. Spain. This work has been partially supported by the Spanish-French Integrated Action no 13 B/93.

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Proceedings of the 5th Eumpeo~~ Conpss on Biotechnology. C. Christiansen, L. Musk and J. Wadsen. eds. vol. l, pp. 233 - 238. Copenhagen: Munksgaard

Mori, T., Nilsson, K., Larsson, P.O. and Mosbacb, K. (1987). Biofechnol. Len., 9.455 - 460. Phillips, R.S., Matte- MS., Olson, E. and von Tersch, R.L. (1990). Ewe Microb. Technol., 12,731 - 735.

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