High yield chromatographic purification of enzymatically produced tyrosine fructosyl ester

Abstract

The synthesis of L L-tyrosine fructosyl ester, from fructose and L L-tyrosine methyl ester, was carried out by a transesteri®cation reaction catalyzed by a-chymotrypsin in water without any organic cosolvent. The yield was optimized by regulating the pH of the reaction medium and a maximum yield of 63% was obtained.A two-step process of tyrosine fructosyl ester puri®ca-tion was proposed using adsorbent resin and activated carbon. Different elutions and supports easily applicable to industrial process have been studied. Solutions of tyrosine fructosyl ester with a 96% purity and 70% recovery yield were obtained by chromatography on a column of acrylic polyester resins with a solution of 0.5 M NaCl as eluant. IntroductionThe synthesis of amino acid derivatives (such as glycosidic esters of tyrosine) is of considerable value in the food and pharmacological areas [1±2]. In the cosmetic industry, N-L malyl tyrosine is already used in skin tanning and skin protection cosmetics [3]. As in this last approach, the use of hydrolases in the synthesis of molecules offers a broad spectrum of industrial applications.Numerous studies have displayed the ability of proteases to acylate sugar with fatty acids [4±7] or to transfer amino acids onto sugars or polyols [8] in anhydrous solvent (pyridine, DMF). Although yields of synthesis were high, the use of organic solvents limits the exploitation of these reactions in the food industry.The synthesis of amino acid derivatives is rarely carried out in aqueous medium with hydrolases due to a preferential hydrolysis.Nevertheless, the synthesis of saccharide-amino acid conjugates using glycosidases has been demonstrated in aqueous medium [9±10] and recently tyrosine fructosyl ester was synthesized, from fructose and L L-tyrosine methyl ester, by reverse hydrolysis reaction catalyzed by a-chymotrypsin in aqueous buffer without cosolvent [2]. The study of this reaction has shown that many parameters in¯uenced the activities of synthesis and hydrolysis and the yield of transesteri®cation. The increase in fructose concentration enabled (i) the synthesis of tyrosine fructosyl ester to be favoured and (ii) the enzyme and the product to be protected against temperature denaturation. Nevertheless, too high a concentration of fructose (above 50% (w/w)) induced a large rise in the viscosity of the reaction medium and the synthesis rate decreased. High temperatures (up to 60°C) enhanced enzyme activity but dropped the product yield due to the very low thermal stability of tyrosine fructosyl ester.The present work concerns the puri®cation of tyrosine fructosyl ester after optimization of its synthesis. Firstly, an optimization of the synthesis is described with pH regulation of the reaction medium. Secondly, a chromatographic process of tyrosine fructosyl ester puri®cation is proposed, using as stationary phase activated carbon and adsorbent resins, commercially available supports often employed in industrial applications. The mode of elutions and the supports were chosen to allow a low cost proce...