Geert Mannens

Immobilization of glutamate decarboxylase and the preparation of an enzyme column for the synthesis of γ-[13N] aminobutyric acid

Abstract The enzyme glutamate decarboxylase ( l - glutamate 1-carboxy-lyase, EC 4.1.1.15 ) is used for the preparation of 1 μmol γ -[ 13 N ] aminobutyric acid, a radiopharmaceutical designed for positron emission tomography. To obtain a rapid and high-yield synthesis of the labelled compound, glutamate decarboxylase was immobilized on controlled pore silica beads ( pore size 50 nm, particle size 100–200 μm ), which were packed into a column. A screening of different immobilization techniques is described. The selected immobilization procedure was further optimized. The synthesis of γ -[ 13 N ] aminobutyric acid as function of enzyme loading, column dimensions and flow rate was investigated .

Immobilization of acetate kinase and phosphotransacetylase on derivatized glass beads

Abstract The enzymes acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1) and phosphotransacetylase (acetyl coenzyme A: orthophosphate acetyltransferase, EC 2.3.1.8) were separately immobilized onto controlled pore glass CPG and silica beads (pore size 50 nm). Different coupling techniques were screened and immobilized enzymes were subjected to storage stability tests. The selected method, the CPG γ-aminopropyl glutaraldehyde succinate dihydrazide, was further optimized to improve the activity of the enzyme-loaded glass beads.

Direct assay for phosphotransacetylase and acetyl-coenzyme a carboxylase by high-performance liquid chromatography

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.

Assay for acetyl-CoA: arylamine N-acetyltransferase by high-performance liquid chromatography applied to serotonin N-acetylation

A specific assay to measure the activity of the enzyme acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver is described. The assay is based on the HPLC analysis of N-acetylserotonin formed by the enzymatic reaction. A reversed-phase column (Spherisorb 5-microns ODS 2; 150 x 3.2 mm) eluted with 0.1 M sodium acetate (pH 4.75)/methanol (75:25) permits baseline separation of serotonin and N-acetylserotonin within 5.3 min. Several variables on the enzyme reaction were studied to obtain maximum activity. The enzyme is most active in glycine buffer at pH 9.5. The apparent Km value for serotonin (at 0.6 mM CoASAc) is 0.246 mM and 9.9 microM for CoASAc (at 1.5 mM serotonin). To avoid acetyl-CoA or N-acetylserotonin consumption in side-reactions, the enzyme was purified. A two-step purification process (ammonium sulfate fractionation and affinity chromatography on immobilised amethopterin) yielded 60-70% of the initial enzyme activity with a purification factor of 455-560.

Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (bakers yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.

Purification and immobilization of acetate kinase from Desulfovibrio vulgaris

SummaryThe enzyme acetate kinase (EC 2.7.2.1) was purified fromDesulfovibrio vulgaris by a combination of ammonium sulfate precipitation, hydroxyl-apatite and dye-affinity chromatography. An overall-purification factor of 15 was obtained resulting in a specific activity of 24 U/mg protein. The purified enzyme was immobilized on differently derivatized controlled pore glass beads.

Immobilization of acetyl-CoA:arylamine N-acetyltransferase and the preparation of an enzyme reactor for the synthesis of N-[11C]acetylserotonin

The enzyme arylamine acetyltransferase (acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) from pigeon liver is immobilized onto differently derivatized controlled pore glass beads. Different silanes, spacer arms and reactive end-groups were tested, and immobilized enzyme stability tests were performed. From these experiments, the method of choice was selected: immobilization on controlled pore glass beads (24 nm pore size, 75-125 microns particle size) derivatized with gamma-aminopropyl and glutaraldehyde as the reactive end group. The kinetic properties of an enzyme reactor were investigated and optimized. The goal was to obtain a rapid high-yield conversion of 0.5-1 mumol acetyl-CoA to N-acetylserotonin, so that the reactor is useful for the 11C-labelling of N-acetylserotonin. Using an enzyme reactor (9.8 x 0.5 cm i.d.) containing 4.6 U active arylamine acetyltransferase immobilized onto 930 mg carrier, a 70% conversion of acetyl-CoA was obtained within 4 min.

Adsorption of charged substrates and products on an enzyme reactor prepared by glutaraldehyde coupling on alkylamine derivatives of Ti(IV)-coated porous silica beads

Abstract Ti(IV) coating of porous silica beads, followed by derivatization with 1,6-diaminohexane and activation with glutaraldehyde was tested for the immobilization of glutamate decarboxylase ( l -glutamate 1-carboxylyase, EC 4.1.1.15). The enzyme column prepared with the immobilized glutamate decarboxylase was designed for the preparation of 1 μmol γ-[13N]aminobutyric acid, a new tracer for positron emission tomography. Preliminary results, indicating high immobilization yields of active enzyme with good long term stabilities, led to a more detailed investigation of the Ti(IV) coating. When a column, containing about 1 g of enzyme-loaded beads was used for the synthesis of γ-[13N]aminobutyric acid (GABA) from l -[13N]glutamate, most of the13N activity remained adsorbed onto the column. The elution patterns of l -glutamate and GABA from columns of glutamate decarboxylase, immobilized on Ti(IV) coated silica beads, were investigated by using an h.p.l.c. u.v. detector. Different treatments of the Ti(IV) coated supports were tested to improve the desorption kinetics of GABA and l -glutamate. None of these methods gave a satisfactory improvement of the elution patterns of GABA and l -glutamate. The results indicate that the Ti(IV) coated silica beads have a large adsorption capacity, even though the enzyme is covalently linked. The described immobilization method is not recommended for enzymes having charged substrates or products and in which a small amount of substrate has to be applied onto a reactor containing a large amount of Ti(IV) coated support. The method can be applied when the enzyme reactor is operated in steady state conditions with continuous supply of substrate.