Activity-guided fractionation of the roots of Anthriscus sylvestris resulted in the isolation and characterization of five cytotoxic compounds, deoxypodophyllotoxin (1), falcarindiol (2), and angeloyl podophyllotoxin (5) from the hexane soluble fraction and morelensin (3), bursehernin (4) from the chloroform soluble fraction. It is the first report of the occurrence of compound 5 in nature.

We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine. Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L-to D-alo-threonine and also from D-to L-alo-threonine. This is the first example of an enzyme that was clearly shown to epimerize threonine. The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000). The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm. The enzyme catalyzes not only epimerization of threonine by stereoconversion at the at position but also racemization of various amino acids, except acidic and aromatic amino acids. The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine.Alanine racemase (EC 5.1.1.1) catalyzes racemization of L-and D-alanine and supplies D-alanine for peptidoglycan synthesis (10). Various other amino acid racemases and epimerases have been demonstrated, but their physiological roles have not yet been clarified. Arginine racemase from Pseudomonas graveolens (11) and amino acid racemase from Aeromonas punctata (6) and Pseudomonas striata (9) show broad substrate specificity but do not act on amino acids such as threonine and valine, whose 3-methylene group is substituted. Amos (2) reported the occurrence of threonine racemase in Escherichia coli. However, it has never been purified or characterized.We 2) containing 10 mM pyridoxal 5'-phosphate was used as the buffer throughout the purification procedure unless otherwise noted. (i) Step 1. The harvested cells were washed twice with 0.85% sodium chloride solution. The washed cells (wet weight, about 1 kg) were suspended in 1 liter of the buffer containing 0.01% 2-mercaptoethanol and disrupted with a sonic oscillator (Kaijo Denki Autochaser 300, Tokyo, Japan) for 30 min. The intact cells and debris were removed by centrifugation. (ii) Step 2. The supernatant solution was brought to 30% saturation with ammonium sulfate and centrifuged at 17,000 x g for 30 min. Ammonium sulfate was added to the supernatant solution to 55% saturation. The precipitate collected by centrifugation was dissolved in the buffer. The enzyme solution was dialyzed against 100 volumes of the buffer.(iii) Step 3. The enzyme solution was applied to a QAE-ZETA prep column that had been equilibrated with the buffer. After the column was washed thoroughly with the buffer containing 20 mM NaCl, the enzyme was eluted with the buffer containing 0.2 M NaCl. The active fractions were pooled, concentrated by addition of ammonium sulfate (65% saturation), and dialyzed against 100 volumes of the b...

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80°C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.Chitosan, a partly acetylated or nonacetylated counterpart (4-linked 2-amino-2-deoxy-␤-D-glucopyranan) of chitin, is present in the mycelial and sporangiophore walls of fungi and the exoskeletons of insects and crustacea (9, 27). It is usually obtained by the artificial deacetylation of chitin in the presence of alkali. Chitosan is a copolymer consisting of ␤-(134)-2-acetamido-D-glucose and ␤-(134)-2-amindo-D-glucose units, with the latter usually exceeding 80% (6). Chitosanase (EC 3.2.1.99) hydrolyzes polymers of (1-4)-␤-D-linked glucosamine (GlcN) residues to chitosan oligomers. Over the last decade, some chitosanolytic enzymes with different substrate specificities have been characterized (9,10,12,25,28,35), and most of them catalyze the endo-type cleavage of chitosan with a narrow range of deacetylation degrees (10,11,26). Recently, chitosan and its partially degraded oligosaccharides have become important because of their potential applications as medical and agricultural agents (2, 36). Thermostable chitosanases active between 60 and 100°C and specifically attacking the ␤-D-glucosaminidic bonds are of special interest (34, 35). Several chitosanases from mesophilic bacteria have been cloned and sequenced to date (1,4,21,22,26). Most of them belong to the thermolabile chitosanases, whereas little information is available on thermostable chitosanases. Thermostability is presumably based on the protein structure. To elucidate the thermostable character of the enzyme, information on its molecular structure, including the entire amino acid sequence and three-dimensional structure, is needed.We have screened bacteria producing thermostable chitosanases and found a strain, Bacillus sp. strain ...

The prevalence of gout is increasing worldwide, and control of serum uric acid level has been regarded as one of the therapeutic methods for gout. Inhibition of xanthine oxidase (XO) activity which can oxidize hypoxanthine to uric acid has been commonly proposed to decrease serum uric acid level. The aim of this study was to demonstrate the hypouricemic effect of ethanol extract of Aster glehni leaves (EAG) by in vitro and in vivo study in potassium oxonate (PO)-induced hyperuricemic rats. EAG possessed 132.5 ± 6.8 mg QE/g of total flavonoid and showed antioxidant activity. EAG showed in vitro and in vivo inhibitory activity against XO and significantly decreased serum uric acid level in PO-induced hyperuricemic rats without liver toxicity. These results show that EAG significantly attenuates hyperuricemia by inhibiting XO activity, which resulted in the decrease of serum uric acid level. Therefore, EAG might possess a potential therapeutic ability for improving gout.