l-Asparaginase II of Saccharomyces cerevisiae

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, highenzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.     

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.     

Production of Enzymes by Plant Cells Immobilized by Sol-Gel Silica

Ajuga reptans cells are cultivated and used for production of invertase. These plant cells are immobilized by a sol-gel SiO₂ membrane, which is built up directly on the cell surface by exposure to a gaseous flow of silicon alcoxide precursors. The immobilization modifies the metabolic activity of cells, resulting in a 40-fold increase in invertase production with respect of free cells. Results concerning total release of proteins, cell growth and produced invertase activity are discussed, considering the absence of breeding, induced by SiO₂ immobilization, the prominent factor promoting the observed exceptional increase in invertase productivity.     

Influence of fadA G203R and ΔflbA mutations on morphology and physiology of submerged Aspergillus nidulans cultures

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans ΔflbA and fadA ^G203R strains were studied. Loss-of-function mutation of the flbA gene resulted in an altered germination with unusually thick germination tubes, “fluffy” pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadA ^G203R mutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the ΔflbA strain, whereas the protease and ammonia production of the fadA ^G203R mutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of γ-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.     

Isolation,characterization, and plasmid pUPI126-mediated indole-3-acetic acid production in Acinetobacter strains from rhizosphere of wheat

Thirty-seven strains of Acinetobacter isolated and characterized from rhizosphere of wheat were screened for indole-3-acetic acid (IAA) production. Only eight Acinetobacter strains showed IAA production. The genus Acinetobacter was confirmed by chromosomal DNA transformation assay. Biotyping of eight strains was carried out and they were found to be genospecies of A. junii, A. baumannii, A. genospecies 3, and A. haemolyticus. Five of eight strains produced IAA at the early stationary phase: A. haemolyticus (A19), A. baumannii (A18, A16, A13), and Acinetobacter genospecies 3 (A15). A. junii A6 showed maximum IAA production at log phase and A. genospecies 3 and A. baumannii (A28, A30) at late stationary phase. IAA was extracted by ethyl acetate and purified by preparative thin-layer chromatography. Purified IAA was confirmed by ¹H-nuclear magnetic resonance and infrared spectrum analysis. Pot experiments showed a significant increase in plant growth inoculated with eight Acinetobacter genospecies as compared to control plants. IAA production was found to be encoded by plasmid pUPI126. All eight strains of Acinetobacter contain a plasmid pUPI126 with a molecular weight of 40 kb. Plasmid pUPI126 was transformed into Escherichia coli HB101 at a frequency of 5 × 10⁻⁵, and E. coli HB101 (pUPI126) transformants also showed IAA activity. PUPI126 also encoded resistance to selenium, tellurium, and lead. This is the first report of plasmid-encoded IAA production in the genus Acinetobacter.     

Invertase production is related to the nitrogen source in Hansenula anomala

Differences in invertase accumulation of Hansenula anomala cultivated in ammonium and nitrate are reported. Media supplemented with sucrose and ammonium sulfateas the solecarbon and nitrogen source, respectively, show strong media acidification (pH 3.0 or lower), and vigorous cell growth. Invertase activity was not detected under such conditions. A cell-free imitation experiment suggests that, under such low pH, extensive chemical break-down of sucrose (>22%) occurs. Thus, H. anomala is able togrow under strong acidic conditions that permit sucrose metabolism by the uptake of monosaccharides generated from chemical hydrolysis. In addition, invertase activity is not present in cells grown in nitrate as nitrogen source at pH 5.0, but at pH 7.0 activity is detected. If ammonium is supplied instead of nitrate, cells grown at pH 5.0 show invertase activity and at pH 7.0 high levels of activity are detected. These results indicate a specific physiological response of the sucrose metabolism to the presence of alternate nitrogen source.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

HPLC Enantioseparation of 1-(α-Aminobenzyl)-2-naphthol and 2-(α-Aminobenzyl)-1-naphthol Analogs on a β-Cyclodextrin-Based Chiral Stationary Phase

The enantiomers of 1-(α-aminobenzyl)-2-naphthol and 2-(α-aminobenzyl)-1-naphthol analogs were separated isothermally on a 3,5-dimethylphenylcarbamoylated β-cyclodextrin-based chiral stationary phase (Cyclobond DMP), with an n-hexane/alcohol modifier as mobile phase. Optimization of the separation was achieved by variation of combinations of the polar mobile phase additives ethanol and methanol. The nature and position of the α-aminobenzyl substituent of the 1- and 2-naphthol analogs influenced the retention and the selectivity.     

The release of heparinase from the periplasmic space of flavobacterium heparinum by three-step osmotic shock

Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure. The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7. Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity. The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material. F. heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.     

Production, purification, and characterization of a polygalacturonase from a new strain of kluyveromyces marxianus isolated from coffee wet-processing wastewater

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M _r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.     

Reactivity of 4-bromomethylquinoline derivatives towards glycine and thioglycolic acid. A new ring system

Condensation of 4-bromomethylquinoline derivatives1 a-1 c with glycine and thioglycolic acid gave the corresponding quinolylmethylglycine and quinolylmethylthioacetic acid derivatives2 a-2 c and2 d-2 f, respectively. Cyclization of2 a-2 f was affected either by polyphosphoric acid or concentrated sulphuric acid to give3 a-3 f. Chlorination of2 a-2 f and3 a-3 f were also accomplished.

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Die Reaktivität von 4-Brommethylchinolin-Derivaten gegenüber Glycin und Thioglycolsäure. Ein neues Ringsystem

Zusammenfassung Die Reaktion von 4-Brommethylchinolin-Derivaten1 a-c mit Glycin und Thioglycolsäure gab die entsprechenden Kondensationsprodukte2 a-c und2d-f.2 a-f konnten mit Polyphosphorsäure bzw. mit konzentrierter Schwefelsäure zu Benzo[c]-2,6-naphthyridinen3 a-f zyklisiert werden.2 a-f und3 a-f waren einer Chlorierung (POCl₃) zugängig.

     

On the interaction of metal extractant reagents. Investigation of the aggregation ofdi(2,4,4-trimethylpentyl)phosphinic acid anddi(n-octyl)phosphinic acid in toluene by vapour-pressure osmometry

Summary The aggregation equilibria ofdi(2,4,4-trimethylpentyl)phosphinic acid (HDTMPP) anddi(n-octyl)phosphinic acid (HDOP) dissolved in toluene have been investigated by vapour-pressure osmometry (VPO) at different temperatures. The experimental data have been treated both graphically and numerically, and the average aggregation numberñ as well as the aggregation constant have been determined. The results suggest that dimers are formed and that the dimerization constant decreases with temperature. Finally, the enthalpies for the aggregation ofdi(2,4,4-trimethylpentyl)phosphinic acid anddi(n-octyl)phosphinic acid in toluene have been estimated using thevan't Hoff equation.

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Über die Wechselwirkung metallextrahierender Reagentien. Untersuchung der Aggregation vondi-(2,2,4-Trimethylpentyl)-phosphinsäure unddi-(n-Octyl)-phosphinsäure in Toluol mittels Dampfdruckosmometrie

Zusammenfassung Die Aggregationsgleichgewichte von in Toluol gelösterdi-(2,4,4-Trimethylphentyl)-phosphinsäure (HDTMPP) unddi-(n-Octyl)-phosphinsäure (HDOP) wurden mittels Dampfdruckosmometrie (VPO) bei verschiedenen Temperaturen untersucht. Die experimentellen Daten wurden sowohl graphisch als auch numerisch ausgewertet; es wurden sowohl die mittlere Aggregationszahlñ als auch die Aggregationskonstante bestimmt. Die Resultate lassen vermuten, daß Dimere gebildet werden und die Dimerisationskonstante mit der Temperatur abnimmt. Mit Hilfe dervan't Hoffschen Gleichung wurden die Enthalpien der Aggregation vondi-(2,4,4-Trimethylpentyl)-phosphinsäure unddi-(n-Octyl)-phosphinsäure in Toluol abgeschätzt.

     

Microcalorimetric Studies on the Promoter Function in E. Coli TG1 from P. Maltophilia AT18 Chromosome DNA

In this paper, a promoter-probe plasmid pKK232-8 was used as a vector, which functioned in Escherichia coli TG₁ host. The plasmid DNA fragments from Pseudomonas maltophilia AT18 chromosome DNA active as promoter inEscherichia coli TG₁, the promoter function was studied by means of microcalorimetry, the promoter is about 800 bp DNA, it can promote the chloramphenicol (Cm) gene in plasmid pKK232-8, the Cm resistance level is about 80 μg mL^–1, the promoter activity is high. It implicates that there are probably many promoters in Pseudomonas maltophilia AT18 chromosome. All these information is readily obtained by an LKB 2277-204 heat conduction microcalorimeter. Microcalorimetry is a quantitative, inexpensive, and versatile method for microbiological genetic research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microbial products. II. Granaticinic acid,a new antibiotic from a thermophilic streptomycete

Streptomyces sp. XT-11989 produces a mixture of two antibiotics with litmus-like indicator properties. One of them was shown to be identical with granaticin, the other was identified as [1S,3S,4S,7R,9R,10S,13R]-4,7,9,10-tetrahydro-5,12-dioxo-4, 6,10,11,13-pentahydroxy-1,9-dimethyl-(1H,3H)-7,10-ethanonaphtho[2,3-c: 6,7-c]dipyran-3-acetic acid and termed granaticinic acid. Microbial production and nuclear magnetic resonance data of these antibiotics are discussed and the antibacterial properties of the antibiotics are compared.

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Mikrobielle produkte. II. Granaticinsäure, ein neues Antibiotikum von einem thermophilen Streptomyceten

Zusammenfassung Der Streptomycetenstamm XT-11989 produziert eine Mischung von zwei Antibiotika mit Lakmus-ähnlichen Indikatoreigenschaften. Eines dieser Antibiotika erwies sich als Granaticin, das andere wurde als [1S,3S,4S,7R,9R,10S,13R]-4,7,9,10-tetrahydro-5,12-dioxo-4,6,10,11,13-pentahydroxy-1,9-dimethyl-(1H,3H)-7,10-ethanonaphto[2,3-c: 6,7-c]dipyran-3-essigsäure identifiziert und Granaticinsäure benannt. Mikrobiologische Produktion und Kernresonanzdaten dieser antibiotika werden besprochen und ihre antibakteriellen Eigenschaften verglichen.

     

Expression and purification of a mutant of human interleukin-2 in Pichia pastoris

Interleukin (IL)-2 is a pharmacologically important cytokine secreted by T-lymphocytes. Recombinant IL-2 (rIL-2) has been modified and produced in many systems. Mass production of rIL-2 is the prerequisite for its wide application. Using a site-directed mutagenesis strategy, we first generated a gene coding for a new type of mutant of human IL-2 (MhIL-2), in which we replaced the cysteine-125 in human IL-2 with alanine, the leucine-18 with methionine, and the leucine-19 with serine. Then we investigated the possibility of its production of MhIL-2 in a Pichia pastoris system. High-level secreted expression of MhIL-2 was achieved by methanol induction. When purified with ultrafiltration, cation-exchange chromatography, and Sephadex G100 gel filtration, about 100 mg of MhIL-2 with high purity was obtained from 1 L of ferment supernatant. Biologic activity assay revealed that the purified recombinant protein displayed increased activity on proliferation of IL-2-dependent CTLL-2 cells. These results suggest that MhIL-2 is an improved IL-2 mutant that might hold great promise for clinical use, and that P. pastoris is an excellent system for the mass production of biologically active hIL-2.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.     

固体表面特征对脲变α-糜蛋白酶折叠的贡献

以脲变α-糜蛋白酶(α-Chy)为模型蛋白, 用蛋白折叠液相色谱法研究了该蛋白在7种不同固体表面上的折叠及其在折叠过程中形成的中间体, 选用疏水相互作用色谱(HPHIC)固定相为吸附剂, 在动态条件下着重研究了疏水色谱固定相TSK和PEG-600表面对脲变α-Chy复性效率的贡献. 用基质辅助激光解吸附离子化飞行时间质谱对3.0 mol•L^－1脲变α-Chy, 在经 HPHIC柱复性并同时分离的收集组分进行确认后, 仅有一种稳定的脲变α-Chy折叠中间体. 发现PEG-600固定相表面较TSK固定相对α-Chy复性效果好. 证实了疏水性强度及固体表面配基的结构对蛋白折叠起着关键性的作用.     

Study on the Chromatographic Behavior of Bases on Dodecylamine-N,N-dimethylenephosphonic Acid-Modified Zirconia Stationary Phase

A new zirconia-based stationary phase (DPZ) was prepared by modifying zirconia with dodecylamine-N,N-dimethylenephosphonic acid (DDPA). DDPA was adsorbed on zirconia with only one phosphonic group, with the other being free. Besides the hydrophobic interaction provided by nonpolar dodecyl, DPZ also has dipolar interaction, ion-exchange or electrostatic repellent interaction provided by the free phosphonic group and amino group at different conditions. Separation of bases on this stationary phase was achieved with satisfaction owing to the various retention mechanisms. The influence of methanol content, pH value, ion types, and ionic strength of mobile phase are studied in detail. Translated from Chinese Journal of Chromatography, 2005, 23(1) (in Chinese)     

Improvement of inulin hydrolysis yeast cell reactor by mutants selection

In a previous publication, we described a continuous production ofd-fructose from enzymatic hydrolysis of inulin with immobilized permeabilized cells (1).Kluyveromyces fragilis ATCC 12424 have been shown to possess inulase activity. The half life of the reactor was at least 1300 h, but productivity was relatively low (around 40 g/L/day). A selection of 50 mutants was tested on liquid medium for a possible increase of productivity. In relation to the improvement of the reactor, the most important factor is intracellular inulase activity, and this activity was increased with the KF 28 mutant. Productivity reached 2000 g/L/day with the increase (of the productivity), proportional to the increase of intracellular inulase activity.