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.     

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.     

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.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

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.     

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.     

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.     

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.     

Strategy and Design of Novel Reagents for the Fluorometric Analysis of Biomolecules

This article covers molecular designs to develop several new fluorometric reagents and their applications to increase the sensitivities up to the picomole level using HPLC for the measurement of biomolecules. The methods were designed to demonstrate the physiological activities, for example (1) N-(9-acridinyl)maleimide (NAM) for the measurement of SH, –S–S–, and sulfite such as cysteine, (2) diphenyl-1-pyrenylphosphine (DPPP) for the hydroperoxides in lipids, serum, tissues, and foodstuffs, (3) 9-bromomethylacridine (9-BrMA), (4) 2-(anthracene-2,3-dicarboxylimide)ethyltrifluoromethane sulfonate (AE-OTf) for carboxylic acids, and (5) The chiral fluorometric labelling reagent (S)-( + )-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid (TBMB) to identify the chiralities of amino acids, sugars, and mono- and diacylglycerols.     

Physicochemical Studies on Sulfated Polysaccharides Extracted from Seaweeds at Various Temperatures

Sulfated polysaccharides (SPS) were extracted from three species of seaweeds of Ulvacea (Ulva pertusa, Ulva conglobata and Entromorpha prolifera) for 4 hr at various temperatures and their physicochemical properties were studied using viscometric and equilibrium sedimentation measurements in order to determine the optimum extracting condition.

Sulfated polysaccharides extracted at various temperatures from the seaweed of U. pertusa had the same physicochemical properties, while the larger molecular components of SPS was not extracted from U. conglobata and E. prolifera, at the low temperature of 30~40°C. This was confirmed by analyses of their viscosity and molecular weight and by gel filtration chromatography, in which each SPS showed two or three peaks.

The larger molecular component of SPS could be extracted at the high temperature of 80~90°C in the thermostable form.     

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.     

Molecular simulation of solid–solid phase transitions

Applications of dl_poly to solid–solid phase transitions are reviewed, with particular attention to how details of the mechanisms of the transitions may be extracted from molecular dynamics simulations. Two examples in molecular crystals are discussed: the order–disorder transition of p-terphenyl initiated at around 200 K by the unlocking of ring flipping; and the rotator phases of n-alkanes with around 20 carbon atoms per chain, showing distinct molecular mechanisms in the dynamics just below the melting points of odd and even chains. Covalent-ionic materials are represented by an application to an aluminophophate molecular sieve, AlPO₄-5.     

Structural Analysis of the Fundamental Polymer of the Sheath Constructed by Sphaerotilus natans

The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing β-D-GlcA, β-D-Glc, α-D-GalN, and β-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

Identity of Brusatol and Yatansin,An Antidysentric Agent

In order to develop a fermentation process for lactase (β-d-galactosidase) production, we selected an excellent lactase producer, Kluyveromyces lactis. KY5466, from our yeast culture collection. Some of its mutant derivatives which formed a blue pigment from 5-bromo-4-chloro-3-indolyl-β-d-galactoside in the presence of glucose and those which assimilated phenyl-β-d-galactoside as a carbon source produced 2 to 2.7 times as much lactase as the parent strain. In the late stage of cultivation, the lactase activity decreased to zero for all strains tested soon after the complete consumption of sugar. This phenomenon was found to be correlated with a decrease in the efficiency of protein extraction from the cells. The maximal amount of lactase produced reached 155 units per ml at 48 hr in a 5-1 jar fermentor culture with sugar feeding.     

Purification and Characterization of β-D-Glucosidase (β-D-Fucosidase) from Bifidobacterium breve clb Acclimated to Cellobiose

The β-d-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β- d-glucosidase activity but also β- d-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000–48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-fucoside were 1.3mm and 0.7 mm, respectively. This enzyme had also transferase activity for the β-d-fucosyl group but not for the β-d-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-d-glucosidase from other bacteria, actinomycetes, and plants.     

Mutant of Escherichia coli Tolerant to the Lysis Induced by Cephalexin

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

Neuroprotection by mefenamic acid against d-serine: Involvement of oxidative stress,inflammation and apoptosis

Abstract

Mefenamic acid, a non-steroidal antiinflammatory drug (NSAID), directly and dose-dependently exhibits neuroprotective activity. In our study, we investigated the effects of mefenamic acid against d-serine on oxidative stress in the hippocampus, cortex and cerebellum of rats. Furthermore, the potential inflammatory and apoptotic effects of d-serine and potential protective effect of mefenamic acid were determined at mRNA and protein levels of TNF-α, IL-1β, Bcl-2 and Bax. We found that d-serine significantly increased oxidative stress, levels of inflammation- and apoptosis-related molecules in a region specific manner. Mefenamic acid treatment provided significant protection against the elevation of lipid peroxidation, protein oxidation, levels of TNF-α, IL-1β and Bax. As a conclusion, we suggest that d-serine, as a potential neurodegenerative agent, may have a pivotal role in the regulation of oxidative stress, inflammation and apoptosis; and NSAIDs, such as mefenamic acid, may assist other therapeutics in treating disorders where d-serine-induced neurotoxic mechanisms are involved in.     

Biosynthesis of Grayanotoxins in Leucothoe grayana Max.Incorporation of Mevalonic Acid and (—)-Kaurene into Grayanotoxin-III

The main subunits of glutenin were separated by preparative SDS-PAGE with a Laemmli system (U. K. Laemmli, Nature, 227, 680 (1970)) and their cysteine (Cys) contents were determined by amino acid analysis. Amino acid compositions of glutenin subunits, determined in the present study, were different from those determined by Danno et al. [G. Danno, K. Kanazawa and M. Natake, Agric. Biol. Chem., 40, 739 (1976)]. We found that these differences were due to the different methods of hydrolysis of subunit polypeptides. That is, hydrolysis of subunit polypeptides extracted from gel and hydrolysis of polypeptides in gel without extraction. Cys contents of glutenin subunits were determined as S-pyridylethyl cysteine (PE-Cys). Although no PE-Cys was detected in B-4 or B-4′, all other subunits were shown to have 4mol Cys per mol protein, respectively.