Seiichiro Ikeda

Preparation of pyridoxal 5′-phosphate-bound Sepharose and its use for immobilization of tryptophanase

Abstract Pyridoxal 5′-phosphate-bound Sepharose (SP) was prepared by coupling pyridoxal 5′-phosphate (PLP) to diazotized p-aminobenzamidohexyl-Sepharose. A derivative of pyridoxine having an absorption maximum at ca. 316 nm (possibly, 6-amino-pyridoxine 5′-phosphate) was liberated from SP by treatment with 0.1 M sodium dithionite at pH 9.0. SP catalyzed the cleavage of tryptophan in the presence of Cu2+, a typical non-enzymatic model of tryptophanase reaction. From the spectrophotometric data and catalytic activity, it was estimated that SP contained about 1.5 μmoles of bound PLP per gram of Sepharose. Tetrameric apotryptophanase was immobilized by incubation with SP, followed by reduction with NaBH4. The resulting immobilized tryptophanase retained ca. 60 % of the catalytic activity of free tryptophanase used. This method was much superior to other methods used commonly for preparation of immobilized enzymes.

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

SummaryProduction 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.

Preparation and characterization of several new immobilized derivatives of pyridoxal 5′-phosphate

Abstract Two types of new Sepharose-bound pyridoxal 5′-phosphate, N-immobilized and 3-0-immobilized pyridoxal 5′-phosphate analogues, were prepared by reacting pyridoxal 5′-phosphate with a bromoacetyl derivative of Sepharose 4B in dimethylformamide (50% v/v) and in potassium phosphate buffer (pH 6.0) for approx. 70 h at room temperature in the dark, respectively. The properties of these immobilized pyridoxal 5′-phosphate derivatives including their catalytic activities in the non-enzymatic cleavage reaction of tryptophan were studied in comparison with those of the 6-immobilized pyridoxal 5′-phosphate analogue reported previously by the present authors. The usefulness of these pyridoxal 5′-phosphate analogues in the preparation of immobilized tryptophanase was demonstrated.

Application of immobilized aspartate aminotransferase and immobilized aspartate aminotransferase—malate dehydrogenase coupled system to micro-assay of L-aspartic acid

Usefulness of immobilized enzymes in analyses has been well recognized [ 1,2] . Especially, immobilized multi-enzyme systems would be the most promising materials for micro-analyses of various biological compounds. Newirth et al. [3] have reported the determination of phosphoenolpyruvate by use of the combined system of immobilized pyruvate kinase and immobilized lactate dehydrogenase. Automatic determination of disaccharides has been performed by Inman and Hornby with some immobilized linked enzyme systems [4]. In previous papers [5,6], the present authors reported the preparation of immobilized tryptophanase and immobilized tryptophanase-lactate dehydrogenase coupled system and the application to the micro-assay of L-tryptophan. This paper describes the immobilization of aspartate aminotransferase (AAT) and of a coupled system of AAT and malate dehydrogenase (MDH) and their application to a micro-assay of L-aspartate.

Affinity chromatography of amine oxidase from Aspergillus niger

Omega-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of epsilon-aminopentyl-Sepharose or delta-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 A) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to omega-aminohexyl-Sepharose. It was also demonstrated that the affinity chromatography on omega-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.

Immobilized derivative of pyridoxal 5'-phosphate. Application to affinity chromatography of tryptophanase and tyrosine phenol-lyase.

Hitherto we have prepared three kinds of immobilized derivatives of pyridoxal-P**: 6-immobilized, N-immobilized and 3-O-immobilized analogues bound covalently to Sepharose4B matrix at the indicated position [ 1,2] . Since all of these Sepharose-bound pyridoxal-P possess free 4-formyl groups and 5’-phosphate groups which are essential for binding with apoproteins of various vitamin B6 enzymes, these compounds are expected to be useful for affinity chromatography and studies on the coenzyme binding properties of vitamin Be enzymes. Immobilization of some vitamin B6 enzymes consisting of multisubunits, such as tryptophanase, was successfully achieved through the specific binding of immobilized pyridoxal-P to the lysine residue at the active site of the one subunit [l-4] . In the present paper we describe the application of one of the abovementioned immobilized derivatives of pyridoxal-P, i. e., 3-O-immobilized pyridoxal-P (referred to as PLP-Seph.) for the affinity chromatography of apotryptophanase from Escherichia coli. The enzyme was efficiently adsorbed on a column of PLP-Seph. and eluted selectively with a mixture of 0.5 M potassium phosphate buffer and 0.5 mM pyridoxal-P. Moreover, PLP-Seph. served as an excellent biospecific adsorbent for apo-tyrosine phenol-lyase requiring pyridoxal-P. These results indicate that Sepharose-bound

Immobilization of Coenzyme B6 and Several B6 Enzymes: Application to Assay or Production of Some Amino Acids and to Structure-Function Interrelationship Studies of B6 Enzymes

Although a number of papers have been published concerning immobilization of various enzymes, little has been reported with respect to immobilization of enzymes requiring coenzymes. This paper describes immobilization of coenzyme B6 (pyridoxal 5’-phosphate, PLP) and several B6 enzymes which require PLP as coenzyme and catalyze a variety of reactions of amino acids. Applications of the immobilized B6 enzymes to production or assay of some amino acids and to studies on the structure-function interrelationship are comprehensively mentioned.