John E. Gavagan

Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W

A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

Abstract A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C3–C6 dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746.

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.

Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D

The genes encoding a thermally stable and regio-selective nitrile hydratase (NHase) and an amidase from Comamonas testosteroni 5-MGAM-4D have been cloned and sequenced, and active NHase has been over-produced in Escherichia coli. Maximal activity requires co-expression of a small open reading frame immediately downstream from the NHase beta subunit gene. Compared to the native organism, the E. coli biocatalyst has nearly threefold more NHase activity on a dry cell weight basis, and this activity is significantly more thermally stable. In addition, this biocatalyst converts a wide spectrum of nitrile substrates to the corresponding amides. Such versatility and robustness are desirable attributes of a biocatalyst intended for use in commercial applications.

Chemoenzymatic production of 1,5-dimethyl-2-piperidone

Abstract A chemoenzymatic process for the preparation of 1,5-dimethyl-2-piperidone (1,5-DMPD) from 2-methylglutaronitrile (MGN) has been demonstrated. MGN was first hydrolyzed to 4-cyanopentanoic acid (4-CPA) ammonium salt using the nitrilase activity of immobilized Acidovorax facilis 72W cells. The hydrolysis reaction produced 4-CPA ammonium salt with greater than 98% regioselectivity at 100% conversion, and at concentrations of 170–210 g 4-CPA/l. Catalyst productivities of at least 1000 g 4-CPA/g dry cell weight (dcw) of immobilized cells were achieved by recycling the immobilized-cell catalyst in consecutive stirred-batch reactions. After recovery of the immobilized cell catalyst for reuse, the 4-CPA ammonium salt in the aqueous product mixture was directly converted to 1,5-DMPD by low-pressure catalytic hydrogenation in the presence of added methylamine.

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.

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.