Stimulation of L-asparate beta-decarboxylase formation by L-glutamate in Pseudomonas dacunhae and Improved production of L-alanine

The formation of L-asparate beta-decarboxylase by Pseudomonas dacunhae was compared on media containing a variety of organic acids and amino acids as a carbon source. Although the enzyme was formed constitutively when the organism was grown on basal medium or on that containing tricarboxylic acid cycle intermediates, it was induced twofold by L-glutamate and repressed one-tenth by L-serine. L-Glutamine, L-proline, L-leucine, glycine, and L-threonine also showed induction effects lower than that of L-glutamate. L-Glutamate derepressed the serine effect. This glutamate effect was observed effect was observed with other microoganisms, e.g., Achromobacter pestifer and Achromobacter liquidum. Since the intermediates from L-glutamate metabolism had no effect, this induction effect was specific to L-glutamate. The formation of some glutamate-related enzymes was measured and is discussed in relation to the formation of L-asparate beta-decarboxylase. L-Asparate beta-decarboxylase was purified to an electrophoretically homogenous state from L-glutamate-grown cells of P. dacunhae, and some properties were compared with those of the enzyme from fumarate-grown cells. The two enzymes were identical in disc electrophoresis, molecular weight, and some enzymatic properties. The industrial production of L-alanine from L-aspartic acid acid was improved by using the culture broth with highly induced L-asparate beta-decarboxylase (9.4 U/ml of broth).     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate beta-Decarboxylase Activity and Application to l-Alanine Production

Whole cells of Pseudomonas dacunhae containing l-aspartate beta-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate beta-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as much as 100% when cell lysis was allowed to occur before immobilization. The pH and temperature optima were determined to be 5.5 and 45 degrees C, respectively. Immobilized P. dacunhael-aspartate beta-decarboxylase activity was stabilized by the addition of 0.1 mM pyridoxal-5-phosphate and 0.1 mM alpha-ketoglutaric acid to a 1.7 M ammonium aspartate (pH 5.5) substrate solution. Under conditions of semicontinuous use in a batch reactor, a 2.5% loss in immobilized l-aspartate beta-decarboxylase activity was observed over a 31-day period.     

Pyocyanine production by Pseudomonas aeruginosa.

Dextrose enhanced the growth of P. aeruginosa but suppressed the biosynthesis of pyocyanine. The preformed pigment could be released from dead cells. Pigmentation was not correlated directly with number of viable organisms in the culture. High concentration of maltose likewise inhibited pyocyanine production. Maltose contained in medium used for pyocyanine production by P. aeruginosa should be kept in low concentration or omitted.     

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.     

Improved large-scale production of vitamin B12 by Pseudomonas denitrificans with betaine feeding

The strategy of betaine control for vitamin B(12) large-scale fermentation by Pseudomonas denitrificans was investigated in this paper. The results obtained in shake-flask experiments demonstrated that betaine could greatly stimulate vitamin B(12) biosynthesis but had an inhibition to cell growth. Based on the influence of betaine on the fermentation of P. denitrificans, betaine feeding was a beneficial strategy to solve the inconsistency between cell growth and vitamin B(12) production. As a result, an effective and economical strategy of betaine feeding was established for vitamin B(12) fermentation in 120-m(3) fermenter, in which betaine was continuously fed to maintain betaine concentration of the broth at the range of 5-7g/l during 50-140h of fermentation.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Enhancement of L-glutamate oxidase production in liquid fermentation of Streptomyces sp. by calcium addition

Summary The effect of calcium ions on production of extracellular L-glutamate oxidase (GluOx) in liquid fermentation of Streptomyces sp. N1 was investigated. By supplementing a relatively large amount of Ca²⁺ (15–40 mM) to the medium, the GluOx production was significantly enhanced, although the microbial growth was inhibited to some degree. For the first time, the highest production (i.e. 6.5 U/ml) and productivity (i.e. 0.25 U/ml/h) of GluOx as ever reported (i.e., 1.2 U/g medium after 144 h solid state fermentation and 0.59 U/ml after 96 h liquid fermentation) were achieved with addition of 30 mM Ca²⁺.     

Stimulation of interleukin-6 production by corticotropin-releasing factor.

Based on the immune-modulating properties of corticotropin-releasing factor (CRF), the effect of this peptide for interleukin-6 (IL-6) production was investigated. Using human peripheral blood mononuclear cells (MNC), the amount of bioactive IL-6 produced was significantly (P less than or equal to 0.05) increased by CRF (10(-10) to 10(-7) M range). However, the IL-6 production of lipopolysaccharide-treated MNC cultures was not modified. At concentrations of greater than or equal to 10 nM, CRF and two analogous peptides (Tyr-CRF and alpha-helical CRF) elicited 16- to 21-fold stimulation of IL-6 production by MNC. Purified monocytes, but not purified lymphocytes, were the cells that responded to CRF action exhibiting nearly 19-fold stimulation at 100 nM concentration. The CRF-induced production of IL-6 cytokine by peripheral blood MNC may suggest a messenger role for this neurohormone in the feedback control of neuroendocrine-immune circuitry.     

L-alanine: 4,5-dioxovalerate aminotransferase from Pseudomonas riboflavina: purification and inactivation by methylglyoxal

L-Alanine:4,5-dioxovalerate aminotransferase, which catalyzes transamination between L-alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from Pseudomonas riboflavina IFO 3140. The enzyme had a molecular weight of 190,000 and consisted of four identical subunits. It was crystallized as pale yellow needles. The enzyme used L-alanine (relative activity 100), beta-alanine (39), and L-ornithine (14) as amino donors. gamma-aminobutyrate (55) and epsilon-aminocaproate (34) were also effective as amino donors. The reaction proceeded according to a ping-pong mechanism and the Km values for L-alanine and 4,5-dioxovalerate were 1.7 and 0.75 mM, respectively. The activity of the enzyme is strongly inhibited by pyruvate, hemin, and methylglyoxal. Methylglyoxal interacted with the enzyme and brought about a complete inactivation.     

Stimulation of tetrapyrrole formation in Rhizobium japonicum by restricted aeration.

Cultures of Rhizobium japonicum were grown with vigorous aeration to stationary phase and were then incubated under restricted aeration for several days. Under these "microaerobic" conditions, cellular heme content increased 10-fold, and visible amounts of porphyrins were released into the culture medium. The two predominant porphyrins produced were identified, on the basis of their spectrophotometric and chromatographic properties, as protoporphyrin and coproporphyrin. The cytochrome complement of microaerobic cells partially resembled that of the symbiotic bacteria in that cytochromes alpha-alpha3 were absent and a CO-binding cytochrome 552 was present. During the period of restricted aeration, at the time that the heme content was increasing, there was a similar 10-fold increase in the activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase. However, during the same period, the activity of succinyl thiokinase (an enzyme that is required in large amounts whether or not heme is being produced) increased only twofold. These results suggest that reduced oxygen tension may play a role in inducing heme synthesis necessary for leghemoglobin formation and bacterial differentiation in soybean root nodules.     

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.