Carbon and nitrogen utilization and acid production by mycelia of the ectomycorrhizal fungusTricholoma bakamatsutake in vitro

Mycelial growth of an isolate ofT. bakamatsutake was tested in media with C/N ratio ranging from 0 to 50 and with 32 carbon and 12 nitrogen sources. The isolate grew best at the C/N ratio of 30. It utilized the monosaccharidesd-glucose,d-mannose, andd-fructose, the disaccharide trehalose, and polysaccharide pectin among the carbon sources; and yeast extract,l-glutamic acid, and ammonium compounds among the nitrogen sources. The growth of ten isolates and secretion of gluconic and oxalic acids were compared ind-glucose, trehalose, and pectin media. The utilization ofd-glucose, trehalose, and pectin differed among the ten isolates, but all the isolates secreted gluconic acid in thed-glucose media and oxalic acid in the pectin media.     

Isolation and characterization of a novel polysaccharide fromBacillus licheniformis NCIB 11634

Summary A polysaccharide producing strain ofBacillus licheniformis was isolated from exudate of raffia palm,Raffia vinifera. The optimum conditions for growth and polysaccharide production have been investigated and established. No appreciable polysaccharide was formed on glucose. It grew best in Czapek-Dox media with sucrose as the carbon source. The polysaccharide has been characterized as a heteropolymer containingd-glucose,d-mannose andd-xylose.     

Glucose isomerase production byStreptomyces sp. CCM 4102

The newly isolatedStreptomyces sp. CCM 4102 strain produced a high level of intracellular glucose isomerase in the media containingd-xylose as inducer of the enzyme, corn-steep liquor, yeast extract and magnesium sulfate. The enzyme synthesis was repressed byd-glucose andd-fructose. The strain did not require cobalt ions for enzyme production.     

Aspergillus niger mutants with increased glucose oxidase production

Summary Aspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain.Paper No. 8393, Nebraska Agricultural Research Division.     

Xylose reductase activity in <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-170 cultivated in semi-synthetic medium and cotton husk hemicellulose hydrolyzate

To develop a new enzymatic xylose-to-xylitol conversion, deeper knowledge on the regulation of xylose reductase (XR) is needed. To this purpose, a new strain of Debaryomyces hansenii (UFV-170), which proved a promising xylitol producer, was cultivated in semi-synthetic media containing different carbon sources, specifically three aldo-hexoses (d-glucose, d-galactose and d-mannose), a keto-hexose (d-fructose), a keto-pentose (d-xylose), three aldo-pentoses (d-arabinose, l-arabinose and d-ribose), three disaccharides (maltose, lactose and sucrose) and a pentitol (xylitol). The best substrate was lactose on which cell concentration reached about 20 g l⁻¹ dry weight (DW), while the highest specific growth rates (0.58–0.61 h⁻¹) were detected on lactose, d-mannose, d-glucose and d-galactose. The highest specific activity of XR (0.24 U mg⁻¹) was obtained in raw extracts of cells grown on d-xylose and harvested in the stationary growth phase. When grown on cotton husk hemicellulose hydrolyzates, cells exhibited XR activities five to seven times higher than on semi-synthetic media.     

Enzymatic properties of the glycine d-alanine aminopeptidase of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.     

Influence of 2,4-d and lactose on pollen embryogenesis in anther culture of potato

Anthers of diploid genotypes of Solanum tuberosum capable of androgenesis were cultured on different media to examine the effect on induction of pollen embryogenesis of 2,4-d and lactose. Anthers cultured in callogenic medium with 2,4-d and sucrose produced pollen derived embryoids only exceptionally. When sucrose was replaced by lactose the frequency of embryogenesis was as high or higher than in embryogenic auxin-free medium. Substitution of lactose for sucrose in the embryogenic medium had no effect. Supplementing the embryogenic medium with 2,4-d strongly reduced the frequency of pollen embryoids in the presence of sucrose but not with lactose.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

Effects of external saccharides on cell wall properties and ion transport inHydrodictyon reticulatum

Various saccharides, when present at osmotically insignificant concentrations in growth media, were tested as to their effects on the cell walls of the green algaHydrodictyon reticulatum, manifesting themselves in differences in cell water and ion contents. Bothd-xylose andd-mannose reduce the cell water content andd-galactose does occasionally the same but onlyd-xylose reduces significantly the intracellular sodium concentration, presumably by forming steric hindrances at the outlets of the sodium pumps at the outer surface of the cell membrane. No significant effects of eitherl-arabinose ord-arabinose on the cell water and ion contents were found.     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Comparative study of chemoattractants for the isolation of motile-spored actinoplanetes

Four chemoattractants and three media were used to isolate actinomycetes from environmental samples. Each chemoattractant was evaluated for its ability to attract motile-spored actinoplanetes. The attractants compared were -collidine,d-xylose, vanillin and phosphate-buffered potassium chloride (bKCl). The method of Hayakawaet al for preparing the chemoattractants was combined with a modified chemotactic method. Of the chemoattractants tested, -collidine yielded a slightly greater number of motile-spored actinoplanetes than bKCl ord-xylose, and a significantly greater number than vanillin. bKCl attracted about the same number of organisms asd-xylose. By using several attractants and media with a variety of soils, distinctly different isolates were obtained with each combination.     

Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

The metabolism of the natural amino acid l-valine, the unnatural amino acids d-valine, and d-, l-phenyglycine (d-, l-PG), and the unnatural amino acid amides d-, l-phenylglycine amide (d-, l-PG-NH₂) and l-valine amide (l-Val-NH₂) was studied in Pseudomonas putida ATCC 12633. The organism possessed constitutive l-amidase activities towards l-PG-NH₂ and l-Val-NH₂, both following the same pattern of expression, suggesting the involvement of similarly regulated enzymes, or a common enzyme. Quite surprisingly, growth in mineral media with l-PG-NH₂ resulted in variable, long lag phases of growth and strongly reduced l-amidase activities. Conversion of d-PG-NH₂ into d-PG and l-PG also occurred and could be attributed to the presence of an inducible d-amidase and the racemization of the amino acid amide in combination with l-amidase activity, respectively. The further degradation of l-PG and d-PG involved constitutive l-PG aminotransferase and inducible d-PG dehydrogenase activities, respectively, both with a high degree of enantioselectivity. Amino acid racemase activity for d- and l-PG was not detected. Correspondence to: L. Dijkhuizen     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Schizosaccharomyces malidevorans sp.n., a yeast decomposing L-malic acid

A new yeastSchizosaccharomyces malidevorans sp.n. is described. It resemblesSchizosaccharomyces pombe but differs from it in appearance of the spores and inability to ferment maltose. It decomposesl-malic acid completely in all grape juice and synthetic media tested, but not thed-isomer. During fermentation a copious evolution of hydrogen sulphide occurs.     

Technical communication: A simple and economically viable medium for the growth of Agrobacterium radiobacter for the production of d-amino acid

A simple and cost effective medium for the growth and d-amino acid production by Agrobacterium radiobacter from hydantoin is reported. The effectiveness of this medium is compared with other synthetic media.     

Study of interaction between calcium and zinc ond-galactose intestinal transport

Zinc is an essential trace element necessary to life. This metal may exert some of its physiological effects by acting directly on cellular membranes, either by altering permeability or by modulating the activity of membrane-bound enzymes. On the other hand, calcium is an essential element in a wide variety of cellular activities. The aim of the present work was to study a possible interaction between zinc and calcium on intestinal transport ofd-galactose in jejunum of rabbit in vitro. In media with Ca²⁺, when ZnCl₂ was present at 0.5 or 1 mM, zinc was found to reduce thed-galactose absorption significantly. In Ca²⁺-free media, where CaCl₂ was omitted and replaced isotonically with choline chloride, the sugar transport was not modified by zinc. Verapamil at 10⁻⁶ M (blocking mainly Ca²⁺ transport) did not modify the inhibitory effect of zinc ond-galactose transport. When 10⁻⁶ M of A 23187 (Ca²⁺-specific ionophore) was added with/without Ca²⁺ to the media, ZnCl₂ produced no change in sugar transport. These results could suggest a possible interaction of calcium and zinc for the same chemical groups of membrane, which could affect the intestinal absorption of sugars.     

d-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli

Recently, we reported on the construction of a whole-cell biotransformation system in Escherichia coli for the production of d-mannitol from d-fructose (Kaup B, Bringer-Meyer S, Sahm H (2004) Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation. Appl Microbiol Biotechnol 64:333–339). Supplementation of this strain with extracellular glucose isomerase resulted in the formation of 800 mM d-mannitol from 1,000 mM d-glucose. Co-expression of the xylA gene of E. coli in the biotransformation strain resulted in a d-mannitol concentration of 420 mM from 1,000 mM d-glucose. This is the first example of conversion of d-glucose to d-mannitol with direct coupling of a glucose isomerase to the biotransformation system.     

d-Mannitol dehydrogenase and d-mannitol-1-phosphate dehydrogenase in Platymonas subcordiformis: some characteristics and their role in osmotic adaptation

d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD⁺. The K _m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K _m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K _m values of the d-mannitol dehydrogenase were shifted to higher values. The K _m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P d-fructose 6-phosphate - Mes 2-(N-morpholino)ethanesulfonic acid - MT-DH d-mannitol-dehydrogenase - MT1P-DH d-mannitol-1-phosphate dehydrogenase - Pipes 1,4-piperazinediethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k _cat = 2.24 ± 0.23 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 95.6 ± 9.7 mM, K _{m d-glucose} = 80.0 ± 8.5 mM, K _{i l-phenylalanine} = 90.0 ± 9.2 mM, K _{i d-glucose} = 13.6 ± 1.42 mM; CRL: k _cat = 0.51 ± 0.06 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 10.0 ± 0.98 mM, K _{m d-glucose} = 6.0 ± 0.64 mM, K _{i d-glucose} = 8.5 ± 0.81 mM.     

Molecular chaperones facilitate the soluble expression of <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolases in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The overproduction of d-aminoacylase (d-ANase, 233.8 U/mg), N-acyl-d-glutamate amidohydrolase (d-AGase, 38.1 U/mg) or N-acyl-d-aspartate amidohydrolase (d-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. d-ANase (313.3 U/mg) and d-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the d-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the d-AAase gene alone.