Spray dried microbes and methods of preparation and use

The innovation offers spray-dried preparations of germs and methods of using those germs.

 

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BACKGROUND

 

 
Glyoxylic acid along with other keto acids like pyruvic acid are important intermediates in the manufacture of different agrochemicals, pharmaceuticals and fragrances. Typical commercial production of glyoxylic acid applies oxidation chemistryor electrochemistry. Electrochemical manufacture entails either the reduction of oxalic acid or the anodic oxidation of glyoxal to form glyoxylic acid whereas compound oxidation normally requires the oxidation of glyoxal in the existence of a strongacid like HNO.sub.3. A consequence of the commercial processes is the creation of waste streams containing various poisonous acids and heavy metals.
 

 
Glycolate oxidase is an enzyme available from various sources, such as green leafy plants and mammalian cells, which then catalyzes the oxidation of glycolic acid to glyoxylic acid, with the concomitant production of hydrogen peroxide. Forinstance, Tolbert et al., J. Biol. Chem., 181:905 (1949), reported that an enzyme, extracted from tobacco leaves, that divides the oxidation of lactic acid into formic acid and CO.sub.2 through the intermediate creation of glyoxylic acid. The addition ofcertain chemicals, such as ethylenediamine, limited the further oxidation of the intermediate glyoxylic acid. The oxidations were carried out at a pH of about 8, typically using glycolic acid concentrations of about 3-40 mM (millimolar). The best pHfor the glycolate oxidation was reported to be pH 8.9. Oxalic acid (100 mM) was reported to inhibit the catalytic action of this glycolate oxidase. Similarly, Richardson and Tolbert, J. Biol. Chem., 236:1280 (1961), reported that the creation of oxalicacid through the glycolate oxidase-catalyzed oxidation of glycolic acid to glyoxylic acid, using enzymes isolated from tobacco, sugar beet, Swiss chard, spinach, or rat liver. Richardson and Tolbert, J. Biol. Chem., 236:1280 (1961), also showed thatbuffers containing tris(hydroxymethyl)aminomethane (TRIS) inhibited the formation of oxalic acid in the glycolate oxidase catalyzed oxidation of glycolic acid. Clagett et al., J. Biol. Chem., 78:977 (1949) reported that the best pH for its glycolateoxidase catalyzed oxidation of glycolic acid with oxygen was roughly 7.8-8.6, and the optimum temperature was 35.degree.-40. degree. C.
 

 
Recent advances in recombinant DNA technology, together with the isolation of cDNA coding for the spinach glycolate oxidase (see Volokita et al, J. Biol. Chem., 262:15825 (1987)), have allowed for the building of microbial strains which areintended to function as alternative, economical enzyme sources. For instance, yeast provides several benefits to commercial uses over Escherichia coli and other bacteria. Yeast can generally be increased to greater densities than germs and are readilyadaptable to constant fermentation processing. It has been reported, for example, which Pichia pastoris could be increased to mobile densities in excess of 100 g/L (U.S. Pat. No. 4,414,329). Added benefits of yeast hosts incorporate the simple fact that manycritical works of the organism, such as oxidative phosphorylations, are found inside organelles and thus aren’t subjected to the possible deleterious effects of the overexpression of foreign enzymatic products. Furthermore, yeasts appear to becapable of glycosylation of expressed polypeptide products, where such glycosylation is important to the bioactivity of the polypeptide product.
 

 

Zelitch and Ochoa, J. Biol.

Chem., 201:707 (1953), and Robinson et al., J. Biol. Chem., 237:2001 (1962), reported that the creation of formic acid and CO.sub.2 from the spinach glycolate oxidase-catalyzed oxidation of glycolic acid resultedfrom the nonenzymatic reaction of H.sub.2O.sub.2 with glyoxylic acid. They observed the addition of catalase, an enzyme which catalyzes the decomposition of H.sub.2O.sub.2, significantly enhanced the yields of glyoxylic acid by suppressing the formation offormic acid and CO.sub.2. In regards to glyoxylic acid generation in yeast, glyoxylic acid was created when glycolic oxygen and acid have been reacted in an aqueous solution in the presence of aminomethylphosphonic acid and a catalyst which is agenetically-engineered microbial yeast transformant that communicates the enzyme glycolate oxidase from spinach ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15), and catalase (EC 1.11.1.6) (see U.S. Pat. No. 5,693,490).
 

IP reviewed by Plant-Grow agriculture technology news