Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii.

D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. The oraS gene, which encodes a protein of 121 amino acid residues with M(r) 12,800, is situated upstream of the oraE gene, which encodes a protein of 753 amino acid residues with M(r) 82,900. The holoenzyme appears to comprise a alpha(2)beta(2)-heterotetramer. OraS shows no significant homology to other proteins in the Swiss-Prot data base. The deduced amino acid sequence of OraE includes a conserved base-off/histidine-on cobalamin-binding motif, DXHXXG. OraE was expressed in E. coli as inclusion bodies. Refolding experiments on OraE indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate or adenosylcobalamin play important roles in refolding process. The K(m) values for d-ornithine, 5'-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5'-phosphate (PLP) are 44.5 +/- 2.8, 0.43 +/- 0.04, and 1.5 +/- 0.1 microm, respectively; the k(cat) is 6.3 +/- 0.1 s(-1). The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, and D-ornithine being present in the assay; no other cofactors were required. A red-shift in UV-visible absorption spectrum is observed when free adenosylcobinamide is bound by recombinant D-ornithine aminomutase and no significant change in spectrum when free adenosylcobinamide is bound by mutant OraE-H618G, demonstrating that the enzyme binds adenosylcobalamin in base-off/histidine-on mode.

Purification and properties of ornithine racemase from Clostridium sticklandii.

Ornithine racemase has been purified to homogeneity from Clostridium sticklandii, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first racemase known to be highly specific to ornithine. This PLP-dependent enzyme has an M(r) of 92, 000, with a K(m) for L-ornithine of 0.77 +/- 0.05 mM and a k(cat) of 980 +/- 20 s(-1).

Molecular cloning and characterization of fengycin synthetase gene fenB from Bacillus subtilis.

A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed in Escherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25 degrees C, an optimum pH of 4.5, a Km value of 922 microM, and a turnover number of 236 s(-1). FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.

[Isolation of bacteria which could perform nitrification and denitrification simultaneously by gene probe].

The bacteria which could perform nitrification and denitrification simultaneously from nitrogen containing wastes in Taiwan were isolated by using the probes made from random DNA fragments of Thiosphaera pantotropha. Two isolates were identified and named Alcaligenes faecalis subsp. faecalis strain 1 and strain 2 respectively. The effects on nitrification and denitrification by different medium pH, oxygen content, addition of different electron donors or inhibitors were studied. The isolates not only could perform nitrification, but also denitrification even in the presence of oxygen. Potassium cyanide could inhibit denitrification; hydrazine and hydroxyamine could inhibit nitrification. Alcaligenes faecalis subsp. faecalis strain 2 shows better denitrification.

Pollen germination is impeded by tap water.

Pollen germination in vitro is totally inhibited in tobacco (Nicotiana tabacum) and other species if tap water is used to prepare the germination medium. This effect is already fully present if tap water accounts for only 25% of the medium. Furthermore, the pollen grains deteriorate rapidly and the culture medium turns yellowish-brown. The water toxicity is not caused by one or several compounds regularly monitored by the water authorities but can be removed by ion exchange purification. Although the factor(s) responsible for the inhibition were not identified, the study clearly shows the presence of such a contaminant in three different Orange County (Southern California) water wells. The fact that a fundamental botanical process like pollen germination is inhibited by a factor in drinking water not included in water quality control causes some general health concern. In addition, crop yield might be largely reduced if overhead spray irrigation with this water is utilized. The experiments also suggest that pollen germination in vitro could serve as a sensitive and simple bioassay for water quality.

Maloalcoholic fermentation by immobilized Schizosaccharomyces pombe.

Cells of Schizosaccharomyces pombe TMB 1138, which are capable of metabolizing-malate, was immobilized in calcium alginate gel to carry out maloalcoholic fermentation. Four milliliters of cell suspension containing about 2.0 X 10(7) cells were entrapped in 16 ml of sodium alginate solution in order to prepare 2% Na-alginate (w/v) gel bead. After activation by incubating at 28 degrees C for 24 h in grape juice, 300 beads of immobilized cells were inoculated into the fermentation medium. After fermentation was proceeded at 25 or 28 degrees C for 24 h by shaking, it could metabolize L-malate completely and the total acidity was also reduced. Under the same condition for batch fermentation, it was found that the utilization of L-malic acid was over 97% for the first 7 days in fermentation medium, 85% for the first 4 days in grape juice and 87% for the first 4 days in wine. Furthermore, for the continuous fermentation in wine, the conversion of L-malic acid reached 92% in 24 h and could be maintained at 75% in the following 9 days.

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains.

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.

[Extracellular thermostable alpha-amylase from Bacillus stearothermophilus Q8].

The alpha-amylase of Bacillus stearothermophilus Q8, previously isolated in our laboratory, was purified by ammonium sulfate precipitation and CM-sephadex chromatography. The activity the of partial purified alpha-amylase was to be protected by bovine serum albumin Ca2+ and Mg2+. The enzyme showed 100% activity at pH 9.0; 98%, at pH 8.0 and 41%; at pH 10.0. It expressed optimal reaction temperature at 90 degrees C, 81% of the activities remained at 100 degrees C. After 15 min incubation at 100 degrees C with the addition of 10 mM Ca2+, the enzyme only retained 67% activity. The enzyme, however, retained 10% of the maximal activity after 2 h incubation at 90 degrees C, in the absence of substrate and with the addition of Ca2+. Of cations, Na+ at 0.1 and 1 mM, Mn2+ at 0.1 mM showed stimulatory effect; of anions OH-Cl-I-HCO3-NO2-N3- at 10 mM showed stimulatory effect. Addition of urea and KMnO4 resulted in the loss of enzyme activities; however, lower concentration of SDS and Tween 80 afforded protection of the enzyme activities. Galactose and maltose were non-inhibitory for the enzyme activities, while, fructose, mannose, xylose and lactose were slightly inhibitory. The relative hydrolysis sequence of polysaccharides were amylose greater than soluble starch = corn starch greater than glycogen.

Citric acid production using immobilized conidia of Aspergillus niger TMB 2022.

Conidia of Aspergillus niger TMB 2022 were immobilized in calcium alginate for the production of citric acid. A 1-mL conidia suspension containing ca. 2.32 x 10(8) conidia were entrapped into sodium alginate solution in order to prepare 3% Ca-alginate (w/v) gel bead. Immobilized conidia were inoculated into productive medium containing 14% sucrose, 0.25% (NH(4))(2)CO(3), 0.25% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O with addition of 0.06 mg/L CuSO(4).5H(2)O, 0.25 mg/L ZnCl(2), 1.3 mg/L FeCl(3).6H(2)O, pH 3.8, and incubated at 35 degrees C for 13 days by surface culture to produce 61.53 g/L anhydrous citric acid. Under the same conditions with a batchwise culture, it was found that immobilized conidia could maintain a longer period for citric acid production (31 days): over 70 g/L anhydrous citric acid from runs No. 2-4, with the maximum yield for anhydrous citric acid reaching 77.02 g/L for run No. 2. In contrast, free conidia maintained a shorter acid-producing phase, ca. 17 days; the maximum yield for anhydrous citric acid was 71.07 g/L for run No. 2 but dropped quickly as the run number increased.

Cytochemical studies of callus development from microspore in cultured anther of rice.

Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day intervals from 0 to 40 days have been carried out. Glutaradehyde-OsO4-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, lipids and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous lipid in the first few days of culture, are androgenic. Less than 30%, which have many lipid granules and some amorphous lipid, become nutritive microspores. Starch grains also accumulate in these nutritive microspores which degenerate at the stage when the androgenic multicellular microspores are in rapid development. The remaining microspores, which have no or little lipid, degenerate early. At about the 100-cell stage, each multicellular unit consists of two cell types, large and small. The large cells contain abundant amorphous lipid and starch grains which the small ones stain intensely with TBO.Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport lipid and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and leaf-like protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus.

[Extracellular thermostable alpha-amylase from Bacillus licheniformis B7].

A thermostable alpha-amylase producing bacterium has been isolated from soil and identified as Bacillus licheniformis B7. Cultural conditions of this strain for alpha-amylase production were studied. The optimum temperature and incubation period for enzyme production were 50 degrees C and 7 days respectively. Starch with a concentration of 2.5% was the best carbon source for enzyme production. Besides the above conditions, the addition of 0.05% FeCl3 X 6H2O, 0.6% yeast extract, 0.35% peptone, 0.2% Na-citrate and 0.008% CaCl2 X 2H2O to the growth medium produced maximum enzyme activity. The alpha-amylase produced by this strain showed maximal activity at 90 degrees C and pH 9.0.

[Studies on the factors influencing the digestion of rice straw by Cellulomonas].

A cellulose-decomposing bacterium has been isolated from soil and identified as a member of the genus Cellulomonas. Rice straw which was delignified with 1% (w/w) sodium hydroxide, then treated with 60% (v/v) phosphoric acid to increase the swelling capacity, could be used effectively by cellulase of Cellulomonas. The effects of other conditions such as pH, nitrogen source, substrate concentration and incubation time on the production of filter paper and endoglucanase activity were studied. Maximum production of endoglucanase and filter paper activity occurred at 28 degrees C in 7 days, in the presence of 1% treated rice straw as substrate at an initial pH of 3.0 when NaNO3 was used as source of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appears to be very close to that for Trichoderma reesei grown in the same substrate.

Transport of glycerol by Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the transport of glycerol was shown to be genetically controlled and to be dependent on induction by glycerol. Accumulation of (14)C-glycerol was almost completely absent in uninduced cells and in a transport-negative mutant. Kinetic studies with induced cells suggested that glycerol may be transported by two systems with different affinities for glycerol. Osmotically shocked cells did not transport glycerol, and the supernatant fluid from shocked cells contained glycerol-binding activity demonstrable by equilibrium dialysis. The binding protein was not glycerol kinase. Binding activity was absent in shock fluids from the transport-negative mutant and from uninduced cells. The glycerol-binding protein was partially purified by precipitation with ammonium sulfate. Mild heat treatment completely eliminated the binding activity of shock fluid and of the partially purified protein. Sodium azide and N-ethylmaleimide inhibited both transport by whole cells and binding of glycerol by shock fluid. It is concluded that transport of glycerol by P. aeruginosa involves a binding protein responsible for recognition of glycerol and may occur by facilitated diffusion or active transport. A requirement for energy has not been demonstrated.