A Novel Kauranoid Isolated from Gibberella fujikuroi

A novel arseno-sugar was isolated from the brown alga Sargassum thunbergii. Instead of the dimethylarsinoyl group reported for algal arseno-sugars, this has a tri-methylarsonium group, which is borne by arsenobetaine, a ubiquitous organoarsenic compound in marine animals. This may be an intermediate between arseno-sugars and arsenobetaine.     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Tridec-1-ene-3,5,7,9,11-pentayne,an Ovicidal Substance from Xanthium canadense

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.     

Cloning and Sequencing of the meso-Diaminopimelate-d-dehydrogenase (ddh) Gene of Corynebacterium glutamicum

The ddh gene of Corynebacterium glutamicum encoding raesodiaminopimelate meso-diaminopimelate (meso-DAP)-d-dehydrogenase (DDH) involved in the lysine biosynthesis was cloned in a DAP auxotroph (dapD4) of Escherichia coli by complementation of the DAP auxotroph. Deletion analysis revealed that a ~1.7-kb XhoI-KpnI fragment contained the ddh structure gene. The specific activity of DDH was increased fourteen-fold when C. glutamicum was transformed with a recombinant plasmid harbouring the cloned ddh gene. Furthermore, the ddh gene has been sequenced [S. Ishino et al., Nucleic Acids Res., 15, 3917 (1987)] and some properties of the ddh gene are discussed.     

Substrate Specificity of Thermostable D-Alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 °C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.     

Preparation of Phgsphatioyll2-3Hjinositgl from Yeast Grown in Medium Containing N;Yol2-3Hjinositol

Phosphatidy[2-³]jinositol was prepared from Saccharoniycts cerevisiae (YSC-2), grown in synthetic meaiurn containing myo[2-³H]inositol. Over 44 μCi (or 81 %) of the racio-labeleo inositol was taken up by the organism, with 34 yCi incorporated into phospnatiaylinositol. Upon purification d) silicic acia-meaium pressure liquia chrcnatography (MPLC), a final yield of 24 to 2b μCi of phosphatiayl[2-³h]inositot with a specific radioactivity of 40 ± 10³ apm/nmoie wäs obtained. The purified phosphatiuyl[2-³H] inositol was founo to be a suitable substrate for phospholipase C from human platelets     

Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

Purification and Properties of Two Chitinases from Streptomyces sp. J-13-3

Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45°C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.     

An Improved Synthesis of dl-Histidine

The 5-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-2-deoxystreptamine derivative and its related compounds were synthesized by a modified Königs-Knorr condensation of 3,4-di-O-acetyl-2,6-dideoxy-2-(2′,4′-dinitroanilino)-6-phthalimido-α-d-glucopyranosyl bromide (I) with 4,6-di-O-acetyl-N,N′-dicarbobenzoxy-2-deoxystreptamine (V) and the corresponding streptamine (XI). The aglycons (V) and (XI) were prepared by selective acetylation of the aminocyclitol derivatives by taking advantage of the reactivity difference between the hydroxyl groups at C₅ and C₄ or C₆. The condensed products were converted to N-acetyl derivatives and were shown to have the α-configuration by PMR spectroscopy.     

ESR Studies on Lipid-protein Interaction in Gluten

The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4,4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.     

Mouse AKR1E1 Is an Ortholog of Pig Liver NADPH Dependent 1,5-Anhydro-D-Fructose Reductase

In many organisms, glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is reduced to 1,5-anhydro-D-glucitol (AG). AF reductase, which catalyzes the latter reaction, was purified from pig liver, but mouse ortholog has not yet been reported. In the database, aldo-keto reductase family 1, member E1 (AKR1E1) showed highest homology to pig enzyme. We confirmed that cloned AKR1E1 is mouse ortholog based on enzymatic properties of purified recombinant protein.     

Relation of Cheddar Cheese Ripening to Bacterial Lipolysis

This investigation was undertaken to find the relationship between fat hydrolysis and lipolytic activities of lactic acid bacteria participated in Cheddar cheese ripening. Increases in titratable acidities due to lactic fermentation were completed at early stage of ripening. Ripening indices (ratio of water-soluble nitrogen to total nitrogen) increased rapidly until 90 days and thereafter gradually up to 150 days. Considerable amounts of free fatty acids were released from cheese fat throughout the ripening period. Cheese bacteria were enumerated on the media of tomato-glucose-agar and acetate-agar. About 70% of bacteria isolated from cheese at age of 150 days were classified into Lactobacillus casei and L. plantarum. Lipolytic activities of lactobacilli isolated were detected definitely on double-layered agar plates containing Victoria blue-stained olive oil. Lipase activities were determined in cheese extracts during ripening.     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.

The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.

The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.

Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 days) provided the accumulation of indole on the fibres was avoided.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

Synthesis of 3Z, 6Z-Dienoic Acids

3Z,6Z-Dienoic acids (C₈-C₁₂ and C₁₈) were for the first time synthesized by coupling 2-acetylenic bromides and 2-(3′-butynyloxy)-tetrahydropyrane followed by stereoselective hydrogenation and oxidation.     

2-C-Methyl-d-erythritol. Produced in plants,forms aerosols in the atmosphere. An alternative pathway in isoprenoid biosynthesis

2-C-Methyltetritols, or 2-methyl-1,2,3,4-butanetetraols, which exist as four stereoisomers, are found to be present in the atmosphere above the Amazonian rainforest. 2-C-methyl-d-erythritol was originally isolated from Convolvulus glomeratus and later synthesized enantiomerically pure. It has been claimed that these compounds are produced from isoprene by radical oxidation in the atmosphere. More recently, detailed analysis has shown that the mixture of stereoisomers from forests in both Brazil and Sweden contains unequal amounts of enantiomers. This shows that the oxidation must be due to enzymatic activity in plants. A review of the history of these compounds, synthesis and the significance of stereochemistry is given. Moreover, the significance of 2-C-methyl-d-erythritol for the non-mevalonate route to isoprenoids is briefly discussed.