David Leroy Anton

Pyruvic acid production using methylotrophic yeast transformants as catalyst

Abstract Permeabilized transformants of the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris which express both the glycolate oxidase (( S )-2-hydroxyacid oxidase, EC 1.1.3.15) from spinach and an endogenous catalase (EC 1.11.1.6) have been used as catalysts for the oxidation of l -lactic acid to pyruvic acid. Oxidations of the sodium or ammonium salts of l -lactate at concentrations of up to 1.06 M were run in unbuffered aqueous solution without pH control and with oxygen sparging. The permeabilized transformant catalysts were recovered and recycled in up to 12 consecutive oxidations of the sodium or ammonium salts of 0.50 M l -lactate, where the initial selectivity to pyruvic acid was typically > 98% at 98% conversion of l -lactate. The pyruvic acid salt was readily recovered from unbuffered reaction mixtures in high yield and purity by separation of the catalyst from the reaction mixture, followed by removal of water by evaporation or freeze-drying.

Glyoxylic acid production using immobilized glycolate oxidase and catalase

A variety of methods for the immobilization of glycolate oxidase have been examined for the preparation of a catalyst for the oxidation of glycolic acid to glyoxylic acid. The co-immobilization of glycolate oxidase and catalase on oxirane acrylic beads produced a catalyst which was stable to the reaction conditions used for the oxidation, where glycolic acid and oxygen are reacted in aqueous solution in the presence of the immobilized enzyme catalyst and ethylenediamine. Under optimum reaction conditions, 99% yields of glyoxylic acid were obtained at greater than 99% conversion of glycolic acid, and the recovery and reuse of the co-immobilized enzyme catalyst was demonstrated.

Structural and Mechanistic Properties of E. Coli Adenosylmethionine Decarboxylase

Adenosylmethionine decarboxylase catalyzes one of the first committed steps in polyamine biosynthesis. It is a member of a small class of decarboxylases that use a pyruvovyl prosthetic group rather than the more common pyridoxal cofactor. We have recently shown that AdoMet decarboxylase from E. coli is composed of stoichiometric amounts of two types of subunits; alpha (Mr = 19,000), and beta (Mr = 14,000). The NH2-terminal of the alpha subunit is blocked by the pyruvoyl group and can be sequenced only after reductive amination, which converts this to an alanine residue. The beta subunit, on the other hand, has an unblocked NH2-terminal and sequences normally. The molecular weight of the holoenzyme, estimated by gel filtration, is 136,000 suggesting that the enzyme is an alpha 4 beta 4 octamer. AdoMet decarboxylase undergoes a time dependent inactivation during turnover. The mechanism of this inactivation involves a transamination from the product, decarboxylated AdoMet, and the pyruvoyl group generating an NH2-terminal alanine. The nascent product aldehyde then eliminates methylthioadenosine, resulting in the formation of acrolein, which covalently labels the alpha subunit. How this mechanism may explain AdoMet decarboxylase turned over, and how AdoMet decarboxylase inhibitors can affect its half life will be discussed.

Enzymatic synthesis of cytidine 5'-diphosphate using pyrimidine nucleoside monophosphate kinase

Abstract Nucleoside monophosphate kinase (NMPK, EC 2.7.4.4, from bovine liver or yeast) has been used to prepare cytidine 5′-diphosphate (CDP). The enzyme catalyses the reversible (Keq ≅ 1) reaction of a pyrimidine nucleoside 5′-monophosphate and ATP to produce a nucleoside 5′-diphosphate and ADP. Equilibrium mixtures of CMP, CDP, ADP, and ATP were obtained from the reaction of CMP, ATP, and magnesium chloride with NMPK. The soluble enzyme could be recovered and reused but an enzyme activity half-life of only ca. 2 days was observed. Stabilization of enzyme activity by immobilization via covalent bonding to a carrier surface, and by gel entrapment, was examined. Immobilization yields were optimized by varying protein loading, pH, temperature, ionic strength, type of buffer, and concentration of enzyme substrates. The highest yields of immobilized NMPK activity were obtained by gel entrapment in a poly(acrylamide-co-N-acryloxysuccinimide) gel crosslinked with triethylenetetramine (PAN-500); NMPK immobilized using this method exhibited increased stability of enzyme activity compared to the unimmobilized enzyme.

A rapid nonchromatographic assay for aminopropyltransferases

Aminopropyltransferases are key enzymes in the biosynthesis of the polyamines spermidine and spermine. A procedure is described for assaying these enzymes by differential charcoal adsorption of 14C-labeled decarboxylated adenosylmethionine substrate from the labeled polyamine product. This assay is linear with time and enzyme concentration, and is suitable for use with a variety of amine acceptors. This procedure has the advantage, over those previously used, that it is extremely rapid yet very sensitive.