Redox reactions in hydrocortisone transformation by Arthrobacter globiformis cells

Hydrocortisone and prednisolone transformation by Arthrobacter globiformis cells in aerobic and anaerobic conditions was studied. 3-Ketosteroid-1-en-dehydrogenase activity was shown to be the major factor regulating the direction of transformation. When it is high (aerobic conditions), the end products of hydrocortisone transformation are prednisolone or its 20 beta-hydroxy derivative. The latter is produced via 1-en-dehydrogenation, which is not a limiting stage of the process. Low 3-ketosteroid-1-en-dehydrogenase activity (in the presence of cyanide) or its complete inhibition (strictly anaerobic conditions) result in the direct reduction of 20-keto group of hydrocortisone.     

The influence of β-cyclodextrin on the kinetics of 1-en-dehydrogenation of 6α-methylhydrocortisone by Arthrobacter globiformis cells

β-Cyclodextrin changes the kinetic peculiarities of microbial 1-en-dehydrogenation of 6α-methylhydrocortisone: the reaction rate, degree of conversion and respiratory-chain activity are increased. Respiratory-chain activity in the presence of β-cyclodextrin is increased both in the presence and in the absence of 6α-methylhydrocortisone. A mathematical model is proposed to describe the kinetics of the process. This model suggests the formation of different multi-component complexes consisting of inclusion complexes and the functional unit involving the enzyme and the respiratory chain. All the parameters of the model were estimated by data fitting. In the model framework the formation of multi-component complexes leads to an increase of the maximal reaction rate and to a decrease of substrate inhibition. An approach is suggested for optimisation of 6α-methylhydrocortisone 1-en-dehydrogenation in the presence of β-cyclodextrin. The optimal concentrations of 6α-methylhydrocortisone and β-cyclodextrin have been calculated. Received: 26 September 1996 / Received revision: 16 January 1997 / Accepted: 17 January 1997     

Kinetic mechanism of choline oxidase from Arthrobacter globiformis

Choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. The enzyme is capable of accepting betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to the aldehyde intermediate and of betaine-aldehyde to glycine-betaine. The steady state kinetic mechanism has been determined at pH 7 with choline and betaine-aldehyde as substrate to be sequential, consistent with oxygen reacting with the reduced enzyme before release of betaine-aldehyde or glycine-betaine, respectively. A K(m) value < or =20 microM has been estimated for betaine-aldehyde based on the kinetic pattern with a y-intercept seen in a plot of 1/rate versus 1/[oxygen]. The kinetic data suggest that betaine-aldehyde predominantly remains bound at the active site during turnover of the enzyme with choline. In agreement with such a conclusion, less than 10% betaine-aldehyde has been found in the reaction mixture under enzymatic turnover with saturating concentrations of choline. The k(cat) values were 6.4+/-0.3 and 15.3+/-2.5 s(-1) for choline and betaine-aldehyde, respectively, suggesting that a kinetic step in the oxidation of choline to the aldehyde intermediate must be partially rate-limiting for catalysis. Cleavage of the CH bond of choline as being partially rate-limiting for catalysis is discussed.     

Kinetic model for the regulation of redox reaction in steroid transformation by Arthrobacter globiformis cells

A kinetic model of hydrocortisone transformation was developed in studies of the kinetics of biochemical systems. The regulatory bases of the model are the biosynthesis of steroid-transforming enzymes and their activity, the level of endogenous substrates, the respiratory chain activity, and the initial concentrations of reagents. When compared, the experimental data completely coincide with the results of the computer modeling, the coincidence being not only qualitative but also quantitative. It indicates that the model suggested can be used for further studies of other transformations of steroid compounds, as well as for transformation of steroid compounds under close-to-biotechnological conditions. The results obtained by means of this model permit one to trace in dynamics the behavior of a number of parameters characterizing the process which is very difficult or not feasible to do in a biochemical experiment. The following was shown: (1) the behavior of the respiratory chain (the reversible transition of its oxidized and reduced forms); (2) the change of the transmembrane potential of hydrogen ions within a far larger stretch of time than is feasible to register in a biochemical experiment; (3) the regulation of the activity of 20beta-hydroxysteroid dehydrogenase and 1, 2-reductase not only by the change in the level of endogenous substrates, but also by means of their biosynthesis; and (4) the regulatory role of 3-ketosteroid-1-en-dehydrogenase.     

Production of Riboflavin by Arthrobacter globiformis

S ummary . The yellow pigment excreted by a strain of Arthrobacter globiformis during exponential growth was identified as riboflav in. The amount produced was approximately 5 μ/mg of cells formed (dry weight) in all media tested, rich as well as poor. This value was also found in media containing 1% of Tween So, even though the concentration of internal flavins then was nearly one-half the normal value. Over production of riboflavin in most media was 40-50 fold. The specific growth rate of the riboflavin producer was sevral per cent less than that of a normal strain of the same species.     

Production of d-Tagatose from Dulcitol by Arthrobacter globiformis

A process for the bacterial oxidation of dulcitol to d-tagatose has been developed. The strain Arthrobacter globiformis ST48 used in this fermentation was isolated from soil. The yield of d-tagatose accumulated in the medium from dulcitol was as high as 85%. About 14 g of d-tagatose crystals was isolated from 1 liter of 2% dulcitol medium.     

L-Lysine production by auxotrophic mutants of Arthrobacter globiformis

By mutagenesis with N-methyl N-nitro-N-nitrosoguanidine, in two steps, a number of methionine plus threonine double auxotrophs have been isolated from a glutamate producing Arthrobacter globiformis, excreting L-lysine in good amounts. For the three potent mutants tested the medium of WHITE was adjudged to be the best. Biotin, ammonium chloride and glucose was found to be optimum at 5 μg l^?1, 40 mM and 4% level, respectively. With such optimal C and N source, the strain MT 35 yielded 28.0 g lysine l^?1 of medium in flask culture.     

Reproductive resting forms of Arthrobacter globiformis

Submerged cultures of Arthrobacter globiformis grown in media unbalanced with respect to carbon and nitrogen sources were found to contain cells exhibiting features typical of resting forms: long-term viability, specific ultrastructure, dormant metabolism, and thermoresistance. Such cells were produced not only in the collection strain VKM B-1112, but also in the A. globiformis strains isolated from 2- to 3-million-year-old permafrost sediments.     

Myceloid growth of Arthrobacter globiformis and other Arthrobacter species.

Transitory myceloid growth occurs in certain complex media with Arthrobacter globiformis strain ATCC 8010. This type of growth, however, was not observed in a medium which contained an array of metal ions but did not contain agents able to complex metal ions. Addition of metal-complexing agents to this medium caused an interruption in the life cycle of strain 8010 so that growth occurred only as the myceloid form. It appeared that manganese was the critical metal that was removed by the metal-complexing agents. During growth, the myceloid cells started to fragment, but wall septation was incomplete. A. globiformis strain ATCC 4336 and several other Arthrobacter species and soil isolates, but not Arthrobacter crystallopoietes, responded to metal-complexing agents as did strain 8010. Biotin and vitamin B12 were not involved in this myceloid growth.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

L-serine dehydratase from Arthrobacter globiformis.

1. L-Serine dehydratase (EC 4.2.1.13) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.     

Arthrobacter globiformis and Its Bacteriophage in Soil

Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells.     

The structure of extracellular polysaccharide of Arthrobacter globiformis

An extracellular polysaccharide from Arthrobacter globiformis is composed of N-acetyl-D-glucosamine, N-acetyl-D-fucosamine, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid and O-acetyl groups in the ratio 1:1:2:1. On the basis of solvolysis with anhydrous hydrogen fluoride, which resulted in a tetrasaccharide fragment, and analysis by 1H and 13NMR spectroscopy, it was concluded that the polysaccharide has the following structure: (formula; see text).     

Adaptive characteristics of salt-induced myceloids of Arthrobacter globiformis

When Arthrobacter globiformis is grown in medium containing increased concentrations of NaCl or decreased levels of cations, the bacteria grow as clusters of branching myceloid cells. The sensitivities of salt-induced and citrate-induced myceloids to several environmental stresses were compared to those of normal exponential-phase bacilli and stationary-phase cocci. Salt-induced myceloids were more resistant than normal cells to ultraviolet light or heat shock at 45°C but not to osmotic upshock or pH 4.3; citrate-induced myceloids showed an intermediate rate of heat inactivation. Carbon or nitrogen starvation of myceloids in the absence of added NaCl or citrate led to their division into single cells. Both myceloids and the single cells derived from them were more resistant than normal bacteria to nitrogen starvation. Salt-induced and citrate-induced myceloids showed reduced metabolism of many different carbon compounds in Biolog GP plates. These studies suggest that the formation of multicellular structures by A. globiformis is an adaptive response which increases its potential for survival.     

Regulation of 3-ketosteroid-1-en-dehydrogenase activity of Arthrobacter globiformis cells by a respiratory chain

It has been shown that 3-ketosteroid-1-en-dehydrogenase localized in a cytoplasmic membrane donates reducing equivalents to a respiratory chain directly which passes them over to oxygen. Microbial hydrocortisone oxidation is coupled with energy generation in the form of the H+ transmembrane potential. Electron transfer via a respiratory chain is the limiting stage in the process of hydrocortisone 1-en-dehydrogenation.     

L-Phenylalanine production by double auxotrophic analogue resistant mutants of Arthrobacter globiformis

A number of tryptophan plus tyrosine double auxotrophic mutants isolated by the NTG treatment of a glutamate producing strain of Arthrobacter globiformis were found to excrete phenylalanine in a mineral salt medium. By controlling the pH of the medium to near neutrality, the active growth period could be extended up to 72 h and more phenylalanine was accumulated compared to the unregulated culture where the growth period took up to 48 h. Under optimum culture conditions, the best double auxotroph (TT-39) produced 3 g phenylalanine/l. Further improvement of phenylalanine production has been achieved by the step-by-step isolation of a mutant resistant to the phenylalanine analogues p-fluorophenylalanine (PFP) and β-2-thienylalanine (TA) from the TT-39 strain. Under optimum culture conditions, the best double auxotrophic analogue resistant mutant TT-39 PT^r-21 yielded 8.7 g/l phenylalanine.     

Cyclic tetrasaccharide-synthesizing enzymes from Arthrobacter globiformis A19

A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil. The enzymes, 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity. The molecular and enzymatic properties of IMT from A. globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75. Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B. globisporus. The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A. globiformis A19. The two genes linked together in tandem and formed a gene cluster, ctsYZ. Both of the gene products showed similarities to alpha-glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes. The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria.     

Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis

A bacteriophage which reproduces on Arthrobacter globiformis ATCC 8010 was isolated from soil. This bacteriophage, designated phiAG8010, propagates either in soft agar or broth cultures of the host. Because of a slow adsorption rate, neither the latent period nor burst size was determined. The mature virion belongs to Bradley's group B and exhibits a hexagonal head measuring 69 nm (length) by 60 nm (width) attached to a sheathless tail 120 nm long. The buoyant density of the mature virion is 1.534 g/cm(3). The mature virion contains double-stranded DNA with a buoyant density of 1.722 g/cm(3) (equivalent to 63.3% G + C). Of 14 strains (representing 13 species) of Arthrobacter examined, including A. globiformis ATCC 4336, only A. globiformis ATCC 8010 supported replication of phiAG8010.