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Permeabilized, metabolically-inactive transformants of the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris which contain significant quantities of the enzymes spinach glycolate oxidase ((S)-2-hydroxyacid oxidase, EC 1.1.3.15), Saccharomyces cerevisiae catalase T (EC 1.11.1.6), and endogenous catalase have been used as catalysts for the oxidation of glycolic acid by oxygen to produce glyoxylic acid in aqueous mixtures containing (aminomethyl)phosphonic acid. After separation and recovery of the microbial catalyst from the oxidation product mixture for reuse, the resulting solution of glyoxylic acid and (aminomethyl)phosphonic acid was subsequently hydrogenated with a palladium/carbon catalyst to produce N-(phosphonomethyl)glycine (glyphosate), a broad-spectrum, postemergent herbicide. Complete conversion of (aminomethyl)phosphonic acid in the hydrogenation allowed the use of a simple acid precipitation for isolation of the N-(phosphonomethyl)glycine from the hydrogenation product mixture in ...

The production of glyoxylic acid from glycolic (hydroxyacetic) acid has been demonstrated using the soluble enzymes glycolate oxidase and catalase as catalysts. Catalase was included as cocatalyst to decompose by product hydrogen peroxide, thus limiting peroxide-dependent formate production and glycolate oxidase deactivation. The addition to reaction mixtures of a primary amine capable of forming the hemiaminal or imine of glyoxylate resulted in an increase in the yield of glyoxylate; hemiaminal/imine mixtures of glyoxylate were not as readily oxidized to formate and carbon dioxide by hydrogen peroxide and also limited product inhibition of glycolate oxidase at high glyoxylate concentrations

The production of glyoxylic acid from glycolic (hydroxyacetic) acid has been demonstrated using the soluble enzymes glycolate oxidase and catalase as catalysts. Catalase was included as cocatalyst to decompose by product hydrogen peroxide, thus limiting peroxide-dependent formate production and glycolate oxidase deactivation. The addition to reaction mixtures of a primary amine capable of forming the hemiaminal or imine of glyoxylate resulted in an increase in the yield of glyoxylate; hemiaminal/imine mixtures of glyoxylate were not as readily oxidized to formate and carbon dioxide by hydrogen peroxide and also limited product inhibition of glycolate oxidase at high glyoxylate concentrations

Publisher Summary Numerous clinical studies have demonstrated that the omega-3 fatty acids in fish oil significantly reduce the risk of cardiovascular disease in adults. This chapter discusses the approach to introduce the genes encoding an omega-3 fatty acid biosynthesis pathway into an oleaginous yeast that synthesizes and stores triglycerides as an energy reserve when starved for nitrogen in the presence of an excess carbon source, such as glucose. It explains the development of a clean and sustainable source of omega-3 fatty acids by fermentation, which uses a metabolically engineered strain of the oleaginous yeast Y. lipolytica. While certain strains of Y. lipolytica can accumulate oil up to 40% of the dry cell weight, the only Polyunsaturated Fatty Acid (PUFA) normally synthesized by the organism is Linoleic Acid (LA). Coordinate expression of desaturase genes and elongase genes comprising a “delta6 pathway” was sufficient to demonstrate the synthesis of Eicosapentaenoic Acid (EPA). However, only an integrated strategy, based on the use of strong promoters, an increase in gene copy numbers, the push and pull of carbon into the engineered pathway, and the use of oleaginous condition, resulted in the generation of a high EPA production strain.

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 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.

The permeabilization of Saccharomyces cerevisiae (baker's yeast), either before or after immobilization in polyacrylamide gel (PAG), has been examined as a means to increase the catalase activity of PAG-immobilized yeast cells. Prior permeabilization of the cells resulted in large losses of catalase activity during immobilization, but permeabilization after immobilization produced increases in the catalase activity of yeast/PAG particles. A dependence of the accessible catalase activity on the concentration of polyacrylamide in permeabilized yeast/PAG particles, and on the method of permeabilization of the immobilized cells, was observed. Optimal levels of stable catalase activity (1000–2000 IU/g PAG particles; ca. 5%–10% of total available activity) were obtained by immobilizing yeast cells (0.5 g wet cells/mL gel) in 10% (w/v) PAG, followed by permeabilization of the entrapped cells with either cetyltrimethylammonium bromide, Triton X-100 and one freeze-thaw, or five freeze-thaw cycles. © 1992 John Wiley & Sons, Inc.

In the rat superior mesenteric arteries, the mechanical responses to perivascular nerve stimulation were characterized. The predominant response was contraction mediated by the release of norepinephrine, acting postjunctionally on α1-adrenoceptors. These frequency-dependent contractions were unaffected by the α2-selective adrenoceptor antagonist yohimbine, but were markedly attenuated by clonidine, the α2-selective adrenoceptor agonist. In the presence of prazosin, the α1-selective antagonist, a significant component of the nerve-mediated contraction was still present. At the concentrations used, prazosin, yohimbine, as well as clonidine acted as competitive antagonists of response to exogenous norepinephrine. This differential inhibition of norepinephrine- and nerve-mediated responses suggested the presence of distinct postjunctional adrenoceptors. The effects of clonidine and yohimbine are interpreted to arise from prejunctional modulation of norepinephrine release. In 30 of the 100 vessels studied, there was spontaneous myogenic tone. In these arteries, field stimulation caused frequency- and voltage-dependent relaxations. These responses were neural in origin, dependent on sympathetic nerve activity, but were nonadrenergic and noncholinergic in nature. Naloxone, indomethacin, and substance P inhibited these relaxations with no significant effect on the tone. The opioid agonist, 1–13 dynorphin relaxed these vessels and only naloxone inhibited this response. The effects of these agents were selective against field-stimulated responses since they did not alter the relaxation to the nonspecific agent sodium nitroprusside. These results provide circumstantial evidence for opioid-mediated vascular relaxation that is presynaptically modulated by prostanoids and substance P.