Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of the raw starch-binding domain.

The cyclodextrin glucanotransferase (CGTase) gene of alkalophilic Bacillus sp. A2-5a was cloned and expressed in Bacillus subtilis ANA-1 as a host. The DNA region included an open reading frame encoding a 704-amino-acid polypeptide with a typical raw starch-binding motif in its C-terminal region. The CGTase purified from Bacillus sp. A2-5a bound to raw starch as strongly as porcine pancreas alpha-amylase, as expected from the sequence motif. A chromosomal region (a DNA fragment of about 14.1 kbp) including the CGTase gene was also cloned and the nucleotide sequence was determined. Possible cyclodextrinase and putative cyclodextrin-binding protein genes were found in the flanking region of the CGTase gene, which implied that the novel starch-degradation pathway postulated for a gram-negative bacterium [Klebsiella oxytoca; Fiedler et al. (1996) J Mol Biol 256: 279-291] also exists in a gram-positive bacterium i.e. Bacillus.

A novel acid phosphatase from Aspergillus niger KU-8 that specifically hydrolyzes C-6 phosphate groups of phosphoryl oligosaccharides.

We had analyzed the detailed structures of the phosphoryl oligosaccharide-1 (PO-1) fraction that was the main component of phosphoryl oligosaccharides (POs) prepared from a potato starch hydrolysate. PO-1 fraction was made up of 3-phosphoryl oligosaccharides and 6-phosphoryl oligosaccharides. Aspergillus niger strain KU-8 produced two types of intracellular acid phosphatase (EC 3.1.3.2, ACPase); ACPase I and II. ACPase II preferentially dephosphorylated 6-phosphoryl oligosaccharides rather than 3-phosphoryl oligosaccharides. The molecular weight of the enzyme was estimated as 66 kDa by SDS-polyacrylamide gel electrophoresis and about 260 kDa by gel filtration, implying the active form to be a tetramer. The optimum pH and temperature of the enzyme were 2.0-2.5 and 60 degrees C, respectively. ACPase II was stable below 50 degrees C for 30 min and pH 2.0-10.0 for 60 min. In spite of the strict specificity toward 6-phosphoryl oligosaccharides in the PO-1 fraction, ACPase II was able to hydrolyze Fru-1,6-di-P, ATP, pyrophosphate, and polyphosphate as well as pNPP and Glc-6-P, a broad substrate specificity.

Determination of felodipine enantiomers using chiral stationary phase liquid chromatography and gas chromatography/mass spectrometry, and the study of their pharmacokinetic profiles in human and dog.

A stereoselective and sensitive method for the determination of the enantiomers of felodipine, a dihydropyridine calcium antagonist, has been developed and the pharmacokinetic profiles of the enantiomers comparatively studied after oral administration to dogs and humans. D6-Felodipine, the internal standard, was added to the plasma, extracted with a solvent and then optically resolved into S(-) and R(+) enantiomers on a high performance liquid chromatographic Chiralcel OJ column. Each enantiomer in the effluent was analysed by capillary column gas chromatography/positive ion electron impact mass spectrometry. After oral administration of the felodipine racemate, the Tmax and t1/2 values hardly differed between the two enantiomers in dogs and humans. The Cmax and AUC0-24 h values of the S(-) enantiomer were slightly higher than those of the R(+) enantiomer in humans but the difference between the enantiomers was not significant. These results suggested that there is no large difference in the absorption, distribution and elimination of felodipine enantiomers after oral administration of the racemate in either dog or human.

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus.

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.

Simultaneous determination of oxygen and cadmium in cadmium and cadmium compounds.

Cadmium and its compounds were analysed for oxygen and cadmium by a modification of the Schütze-Unterzaucher method. Oxygen in some compounds such as cadmium oxide, nitrate and sulphate could not be determined by the usual method. The method of adding carbon was employed for the determination of total oxygen. Total oxygen could be determined by the addition of 5 mg of carbon to a sample boat and heating at 950 degrees . The determination was also carried out by addition of naphthalene (2 mg). It was found that the cadmium powder and cadmium flake used contained ca. 1 and 0.15% oxygen, respectively. Oxygen and cadmium in cadmium and its compounds were simultaneously determined by the addition of 2 mg of naphthalene. Cadmium was determined colorimetrically by use of glyoxal-bis-(2-hydroxyanil). Oxygen and cadmium in the samples could be determined simultaneously with an average error of -0.02 and -0.22%, respectively.