Production ofl-tryptophan,l-tyrosine and their analogues by use of immobilized tryptophanase and immobilizedβ-tyrosinase

Abstract

Production of L-tryptophan, L-tyrosine, or their analogues was attempted using immobilized tryptophanase or β-tyrosinase. The immobilized tryptophanase used in this study was first prepared by the present authors by coupling of free apoenzyme fromEscherichia coli B/1t-7A to pyridoxal 5′-phosphate (PLP) previously bound on Sepharose. This immobilization method involves the formation of Schiff base linkage between 4-formyl group of Sepharose-bound PLP and the α-amino group of the lysine residue of the catalytic center of one subunit of tetrameric apotryptophanase, followed by reductive fixation of the Schiff base linkage with NaBH4. In the case of β-tyrosinase fromEscherichia intermedia having two catalytic centers, however, immobilization by direct coupling to CNBr-activated Sepharose or a bromoacetyl derivative of Sepharose was more suitable than by the coupling to Sepharose-bound PLP. In each case, the affinity for substrate or coenzyme was scarcely affected by the immobilization. The immobilized enzymes thus obtained were shown to possess higher thermal stability and higher resistance to denaturing agents than the free counterparts. The optimal temperature for a short time reaction (10 min) was ca. 70°C for immobilized tryptophanase or 55°C for immobilized β-tyrosinase. In each case the optimal reaction temperature mediated by the immobilized enzyme was fairly higher than that catalyzed by the respective free enzyme. Addition of ethanol (5%, V/V) to the reaction mixtures favored the tryptophanase and β-tyrosinase reactions. The equilibrium of α, β-elimination reactions of L-tryptophan and β-tyrosine lied so far to the synthetic side (70% in tryptophanase and 80% in β-tyrosinase reactions, respectively). By continuous flow methods using these immobilized enzyme columns, L-tryptophan, L-tyrosine, and their analogues, such as L-DOPA and L-5-hydroxytryptophan, were conveniently synthesized in good yields.