α-Methylene-γ-butyrolactone (tulipalin A) was isolated and identified as an insecticidal component from thunberg spiraea, Spiraea thunbergii. This compound showed high insecticidal activity against Thrips palmi.
(+)-(2R,3R)-7-Formyl-6-methoxy-2-methoxymethyl-3- (3,4-methylenedioxyphenyl)-2,3-dihydro-1,4-benzodioxin (2), a key building block for the total synthesis of haedoxan A, was synthesized from (4R)-4-(phenylmethyl)-2-oxazolidinone (3) in ten steps with a 12% overall yield.
(-)-Indolactam-V (1) without a hydrophobic chain at positions 6 and 7 of the indole ring is a weak tumor promoter compared with teleocidin Bs. To investigate the effects of the hydrophobic substituent at position 6 of teleocidin Bs, we synthesized (-)-6-n-octyl-indolactam-V (2) by a palladium-catalyzed coupling reaction from (-)-6-bromo-indolactam-V (7) which had been obtained by microbial conversion with Streptoverticillium blastmyceticum NA34-17 as the key step. (-)-7-n-Octyl-indolactam-V (3) with potent biological activities comparable to those of teleocidin Bs was similarly synthesized from (-)-7-bromo-indolactam-V as a positive control. Compound 2 showed similar biological activities to those of 3, indicating that the effect of the hydrophobic substituent at position 6 of 1 was identical to that at position 7.
The substrate specificity was studied for the metabolic degradation of N-acetyl-D-glucosamine (GlcNAc) derivatives by Rhodococcus rhodochrous IFO 15564 which possesses N-acetyl-D-glucosamine deacetylase as a key-step enzyme. This microorganism degraded a wide range of substrates with modified N-acyl groups. The metabolizing activity of this strain became low to the substrates substituted at 1,3,4,6-positions of GlcNAc, and GlcNAc itself was suggested to be metabolized via an open-chain aldehyde form. Based on these results, a simplified procedure for the isolation of allyl α-N-acetyl-D-glucosaminide from an α, β-anomeric mixture was developed by selectively hydrolyzing the β-anomer with Jackbean β-N-acetyl-D-glucosaminidase and subsequently degrading the resulting N-acetyl-D-glucosamine in the reaction mixture with this microorganism.
Pestalotiopsis theae is known to be a causal fungus for tea gray blight disease. Pesthetoxin was isolated from P. theae as a potent leaf-necrosis substance against tea. The structure of pesthetoxin was established principally by NMR studies to be of four different enolic forms, viz the pairs of internal tautomers (1a)/(1b) and (1c)/(1d) with external tautomerism between them.
We developed a practical preparation procedure for K-252a by methylating K-252b on an industrial scale. The water-insoluble K-252a, which was present in the cell mass, was converted to the water-soluble K-252b Na salt in an alkaline solution. The obtained K-252b was methylated with dimethylsulfate in the presence of potassium carbonate in dimethylacetamide. We have already used this method to manufacture 90 kg of K-252b from the fermentation broth, and regenerated 65 kg of K-252a from K-252b.
Recently we found that a single administration of T-2 toxin (T-2), a trichothecene mycotoxin, into mice induced DNA fragmentation, a biochemical hallmark of apoptosis, in the thymus.1) In this study, we investigated the effective chemical structure(s) of T-2-derived metabolites capable of inducing thymic apoptosis in vivo in mice. Metabolic conversion of T-2 to 3′-hydroxy-T-2 toxin (3′-OH-T-2) (Fig. 1) did not diminish the apoptosis-inducing activity, since essentially the same level of fragmented DNA was detected in the thymus taken from mice injected with either T-2 or 3′-OH-T-2. In contrast, hydrolysis of T-2 and 3′-OH-T-2 at the carbon-4 (C-4) position to HT-2 toxin (HT-2) and 3′-hydroxy-HT-2 toxin (3′-OH-HT-2), respectively, greatly decreased the level of DNA fragmentation. Similarly, hydrolysis of T-2 at the carbon-8 (C-8) position to neosolaniol strongly diminished its ability to induce DNA fragmentation. T-2 tetraol, having no ester groups, was unable to induce apoptosis. Based on the data presented in this study, we concluded that both the acetyl group at the C-4 position and the isovaleryl or 3′-hydroxyisovaleryl group at the C-8 position of the T-2 molecule are important for inducing cell death through apoptosis in the thymus.
There are some similar characteristics in protein nature between the lipase inhibitor from soybean seed and soybean lipoxygenase-1 (LOX-1). Thus, the inhibiting protein for pancreatic lipase was prepared from defatted soybean meal by the procedure for the isolation of LOX-1 [Axelrod et al., Methods in Enzymology, 71, 441-451 (1981)]. The LOX-1 from soybean seed dose-dependently inhibited the release of fatty acid from a soybean oil emulsion, and the concentration of LOX-1 to cause half inhibition of the lipase activity was 3.2×10-7 M. The LOX-1 obtained from E. coli transfected with a plasmid carrying the soybean LOX-1 gene also inhibited the lipase activity. However, the lipase-inhibiting activity by the LOX-1 was not affected by the presence of nordihydroguaiaretic acid, an inhibitor for LOX, in the reaction mixture. These results show that the soybean LOX-1 inhibits lipase activity regardless of its lipoxygenase activity.
It was found that both bacteroids and free-living cells of Rhizobium leguminosarum bv. phaseoli were highly susceptible to hydroxylysine (Hyl) and the inhibition of RNA and/or protein synthesis caused by the hydroxyl residue of Hyl appeared to be responsible for the growth inhibition. The size of free-living cells was enlarged by the addition of Hyl and some cells reached around 5 μm, which were close to the length of bacteroids. Under the same condition, the polyhydroxybutyrate (PHB) content in the cells was conspicuously increased. These results suggest that Hyl is not only a notable growth inhibitor of Rhizobium bacteria but also plays a role in a differentiation to bacteroids.
Various derivatives of recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter. A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renaturation process and three-step chromatography. Refolding succeeded in very high yield using a urea system. The purity of KW-2228 was greater than 99% as measured by SDS-PAGE and HPLC analysis. According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations. This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and derivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rhG-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the same conditions. Thus, the four amino acid substitution (Ala1, Thr3, Tyr4, Arg5) of the N-terminal sequence may facilitate crystallization. The change of Cys17 to Ser may not influence the stability and activity of hG-CSF.
We purified and characterized a thermophilic β-galactosidase from Thermus sp. A4 isolated from the Atagawa hot spring (Shizuoka, Japan). The enzyme was monomeric, and its molecular mass was estimated to be 75 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was extremely thermostable and retained its full activity after incubation at 70°C for 20 h. The Km observed were 5.9 mM for o-nitrophenyl β-D-galactopyranoside and 19 mM for lactose. We cloned and analyzed the complete sequence of the gene encoding this enzyme. It was found to consist of 645 amino acid residues. We propose that this enzyme and seven other unclassified β-galactosidases are new members of family 42 of the glycosyl hydrolases.
Xylanase induction by L-sorbose was studied in a resting cell system of a filamentous fungus, Trichoderma reesei PC-3-7, a hypercellulolytic mutant, and compared with that by other inducers. L-Sorbose induced xylanase activity as well as cellulase. It induced a higher level of xylanase activity than sophorose and xylose did. Three main xylanases, xylanase I (Xyn I), xylanase II (Xyn II), and a non-specific endoglucanase I (EG I), were separated using cation-exchange chromatography, and their activity were measured. Xyn II was induced in about the same proportion (60-80% of the total xylanase activity) by all inducers used. On the other hand, Xyn I was apparently induced by L-sorbose, xylose, and xylooligosaccharides, but only a little by sophorose. Northern blot analysis showed that L-sorbose induced Xyn I and Xyn II at the transcriptional level, and more xyn1 mRNA was transcribed after L-sorbose addition than after sophorose. These results suggested that the expressions of both Xyn I and Xyn II are regulated, at least in part, in a different manner. Furthermore, the Xyn I induction by L-sorbose indicated that an unknown common regulatory mechanism may exist between Xyn I and cellulase inductions.
Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4°C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr (XYN3tr) and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from β-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.
The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.
Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.
The transcripts for 38-kDa and 40-kDa cationic peroxidases of tobacco plants are accumulated in cultured cells, cultured roots, and roots of intact plants. In roots, 38-kDa and 40-kDa peroxidases appear to be localized to apoplasts, where peroxidase proteins may possibly be bound to the cell wall.
A mutant angiotensinogen, S14N, in which Ser14 of ovine angiotensinogen was replaced by Asn to form a N-glycosylation site, was produced in CHO cells. The molecular weight was about 3,000 larger than that of wild-type ovine angiotensinogen, indicating that S14N angiotensinogen was glycosylated at Asn14. In the reaction with human renin, the Km of mutant angiotensinogen was 3 times increased, but the Vmax was not affected by the mutation.
The effects of batatasin III and its analogs on gibberellic acid (GA3)-dependent α-amylase induction in embryoless barley seeds and on cress root-growth were examined. Batatasin III was most effective and caused 68% inhibition of α-amylase induction at 4×10-4 M, but its potency was low compared with that of abscisic acid. In the cress test, p-hydroxybibenzyl had high activity.
Six cyclic peptides with 5, 7, 9, 11, 13, and 15 amino acids, with the inhibitory sequence of the α-amylase inhibitor tendamistat, were synthesized. The 11-residue peptide inhibited porcine pancreatic α-amylase most potently (Ki 0.29±0.09 μM). For the 11-residue peptide, the circular dichroism study suggested a preliminary relationship between its inhibitory activity and structural property.
Yeast had been known to contain only one kind of phospholipid deacylating enzyme with an optimal pH in the acidic range. However, among 8 genera and 25 strains, 7 genera and 13 strains had phospholipid deacylating activity at pH 8.0, when screening of enzyme activity was done with micellar and liposomal substrates. The enzymatic properties of phospholipid deacylating enzyme existing in yeast was not related to genetic identity based on 18S rRNA gene sequence. The results suggest that yeast contains enzymes showing a variety of properties depending on the species of yeast, and further studies on this enzyme with more varieties of yeasts are necessary for understanding the physiological roles of the enzyme in yeast.
A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.
Human NP220 (hNP220) is a novel DNA-binding nuclear protein, which has an arginine/serine-rich motif and polypyrimidine tract-binding motif, and NP220s and matrin 3 are thought to form a novel family of nuclear proteins. We have determined a chromosomal localization of the cDNA encoding human NP220 to 2p13.1-p13.2 by using fluorescence in situ hybridization. Human matrin 3 cDNA was mapped to chromosomes 1p13.1-p21.1 and 5q31.3, demonstrating that these novel nuclear proteins with similar functions are on different chromosomes.
The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method. 1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing β anomer formation. 2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.
Cell agglutination in cell suspensions of 10 strains of Clostridium by lactoferrin (Lf) was observed by obtaining the ratio of increased absorbance (RIA) at 450 nm. The RIA values were very different among the species, being higher in the cell suspensions with bovine Lf (bLf) than in those with human Lf. The binding ability of bLf to the bacterial cells was also observed by an enzyme-linked ligand-binding assay, using the conjugate of iron-free or iron-saturated bLf with horseradish peroxidase (HRPO). The binding ability of bLf was very different among the 10 species, and showed a significant correlation with the cell agglutination of each strain. bLf formed a complex with the cells of C. perfringens, did not dissociate in 2 M NaCl or 4 M urea, but did dissociate in 1 M KSCN. These results suggest that the agglutination of cells of the Clostridium species by bLf is probably caused by the cooperative action of at least electrostatic and hydrophobic interactions between bLf and certain components of the cell surface.
The binding characteristics of bovine lactoferrin (bLf) to cells of the Clostridium species were observed by using a horseradish peroxidase-bLf conjugate. A bLf-binding protein (BP) having a relative molecular mass of about 33 kDa was confirmed in the surface layer components from 7 strains of the Clostridium species. The binding of the conjugate to bLf-BP of C. perfringens was strongly blocked by intact Lfs, lysine or arginine residues modified bLf, and deglycosylated bLf, but was not by other milk proteins or by the constituent sugars of glycan. Bacterial growth was inhibited by bLf, but was slightly inhibited by lysine residues modified bLf or deglycosylated bLf. Lactoferricin B did not block the binding of the conjugate, but strongly inhibited the bacterial growth. This suggests that the lysine or arginine residues and glycan of bLf hardly participated in binding bLf to the bacterial cells, but that the amino acid residues and glycan played an important role in inhibiting the growth of bacteria.
Polyphenol extract from barley bran (BPE) induced nitro blue tetrazolium (NBT) reducing activity and alpha-naphthyl butyrate esterase activity in HL60 human myeloid leukemia cells. Because BPE induced the biochemical markers of HL60 cell differentiation, we investigated the effects of proanthocyanidins isolated from BPE on the HL60 cell differentiation of HL60 cells. Prodelphinidin B-3, T1, T2, and T3 induced 26-40% NBT-positive cells and 22-32% alpha-naphthyl butyrate esterase-positive cells. Proanthocyanidins potentiated retinoic acid (all-trans-retinoic acid)-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells.
An easy and highly sensitive method for measuring histamine by HPLC analysis coupled with precolumn derivatization was established. The amino group of histamine was completely colorimetrically labelled with 4-N,N-dimethylamino-azobenzene-4′-isothiocyanate (DABITC) in the presence of sodium bicarbonate at 90°C for 5 min. The derivative was sensitively and easily analyzed by HPLC on a Cosmosil 5SL column using CHCl3/N,N-dimethylformamide/H2O (210:90:4) containing 0.4% acetic acid. Using the established method, histidine decarboxylase (HDC) inhibitory activities of three tartaric acid isoflavone derivatives, named shoyuflavones, isolated from soy sauce were examined in vitro by measuring the histamine produced by HDC. They showed intense inhibition of the activities of HDC from both mouse mastocytoma P-815 cells and Clostridium perfringens.
The effects of dietary oligosaccharides on the hepatotoxic action of D-galactosamine (GalN) were investigated in this study. Male Wistar rats fed with 20% casein diets containing 10% oligosaccharide or D-galactose (Gal) for 2 weeks were injected with GalN (1,900 mg/kg of body weight), and the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the hepatic glycogen concentration were examined 20 hours after the injection. The plasma AST and ALT activities in experiment 1 for the Gal+neomycin (NEO) group were significantly lower than those for the control (C), NEO, raffinose (RAF)+NEO and galacto-oligosaccharide (GALO)+NEO groups. In experiment 2, these activities were significantly lower in the Gal, Gal+NEO and RAF groups than in the RAF+NEO group when the groups were treated with GalN. On the other hand, in respect of the hepatic glycogen concentration in experiment 1, that of the Gal+NEO group was higher than that of the C, NEO, RAF+NEO or GALO+NEO groups. In experiment 2, this parameter was significantly higher in the Gal, Gal+NEO and RAF groups than in the RAF+NEO group after the GalN treatment. As a result, it is suggested that the GalN-hepatitis-suppressive effects of indigestible oligosaccharides such as RAF or GALO is mediated by the action of intestinal bacteria.
The antiulcer activity of cacao liquor water-soluble crude polyphenols (CWSP) was examined. CWSP, α-tocopherol, sucralfate (500 mg/kg), and cimetidine (250 mg/kg) were orally administerted to male SD rats 30 minutes before ethanol treatment. 5 ml/kg of ethanol given intragastrically caused lesions in mucosa of the glandular stomach. CWSP caused a reduction of such hemorrhagic lesions as well as cimetidine and sucralfate which are typical antiulcer drugs, but α-tocopherol was less effective. Thiobarbituric acid reactive substances in gastric mucosa significantly increased with ethanol administration. CWSP treatment significantly reduced this change. The administration of ethanol extensively increased myeloperoxidase (MPO) but not xanthine oxidase (XOD) activity. CWSP reduced the activities of both enzymes; they were considered the main sources of oxygen radicals. According to an in vitro study, CWSP directly reducted XOD but not MPO. These results suggest that the antiulcer mechanism of CWSP was not only radical scavenging but also modulation of leukocyte function.
The plasma level of sialic acid (NeuAc) in inherently scorbutic [Osteogenic Disorder Shionogi (ODS)] rats was increased by 21 days of vitamin C deficiency and simultaneous vitamins C and E deficiency. The brain content of NeuAc was decreased by deficiencies of these vitamins. The NeuAc level in the liver was not affected significantly by these deficiencies.
3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.
We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones. The insert size ranged from 35 to 175 kbp, with an average of 120 kbp. The library is about 18 genomes equivalent, therefore covering more than 99.999% of the genome. This library is the first to be constructed using a rice pathogenic wild type isolate. Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency. Total library clones were arranged onto two nylon membranes for efficient screening. Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions. As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed. Of all the BAC clones, 10%, 13%, 18%, 12%, and 23% contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively. The percentage of clones possessing more than five kinds of transposons was 1.4%, 215 times greater than the expected number. The results show that these transposons were distributed in clusters in the M. grisea genome.
The antimicrobial activities of standard solutions of three organic acids (lactic, acetic, and propionic acids) were compared using Micrococcus luteus, Pseudomonas sp. and Staphylococcus aureus as test microorganisms. At the same concentrations of the undissociated form, the antimicrobial activities of acetic and propionic acids were higher than that of lactic acid, irrespective of test microorganisms. In a single cultivation of Bifidobacterium longum, a mixture of lactic (17 g/l) and acetic (20 g/l) acids was produced from 50 g/l lactose and its antimicrobial activities against M. luteus, Pseudomonas sp., and S. aureus correspond to that of 32, 19, and 25 g/l of acetic acid, respectively. To increase the total antimicrobial activity, a co-culture of B. longum and Propionibacterium freudenreichii, in which lactic acid produced once from lactose by B. longum was converted to acetic and propionic acids by P. freudenreichii, was done using TPY medium containing commercially available peptones as a nitrogen source. By the sequential conversion of lactose using the two microorganisms, the culture supernatant containing a mixture of acetic (27 g/l) and propionic (13 g/l) acids without lactic acid was produced. The antimicrobial activities of the mixture against M. luteus, Pseudomonas sp., and S. aureus were 35, 30, and 26 g/l as a concentration of acetic acid, respectively, higher than that obtained in the cultivation of B. longum alone. When the medium containing an enzymatic hydrolyzate of whey proteins with a protease was used in the co-culture of B. longum and P. freudenreichii, the culture supernatant containing the mixture of organic acids was also obtained in the same manner as the co-culture using TPY medium and the activities were 43, 29, and 29 g/l as a concentration of acetic acid for M. luteus, Pseudomonas sp. and S. aureus, respectively.
A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0, while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1β subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1β subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.
Threonine aldolase activities (threonine acetaldehyde-lyase activity) with different stereospecificities toward 4 isomers of threonine were found in a variety of aerobic bacteria. The strains could be divided into three groups on the basis of the substrate specificity of a cell-free extract. The enzyme activities toward D-allo- and D-threonine were inhibited by EDTA, which suggested that a metal ion participates in the D-specific aldolase reaction.
An enzymatic method for glycolaldehyde production from ethylene glycol was investigated using immobilized alcohol oxidase and catalase. Those enzymes were immobilized onto Chitopearl BCW 3501. When only alcohol oxidase was immobilized onto it, the apparent activity was 190 units/g in wet gel using methanol as the substrate. Tris-HCl buffer (1.5 M; pH 9.0) was selected based on a high stability of glycolaldehyde and a low production of glyoxal as a by-product. Under the optimum conditions, 0.97 M glycolaldehyde was formed from 1.0 M ethylene glycol and the ratio of glyoxal to glycolaldehyde was less than 1%.
The nucleotide sequence of spoIIIE-like and the sporulation-inhibitory and transfer gene (spi) in a conjugative plasmid, pSA1.1, of Streptomyces azureus were examined to detect the promoter region. Using Southern blotting and a spi fragment as probe, spi-like genes were detected in chromosomes of the host and other actinomycetes. These results suggest that there is a spi- and spoIIIE-like gene in chromosomes of some actinomycetes.
Tetragenococcus halophila D10 decarboxylates aspartate to alanine, but T. halophila D10 derivatives generated by a curing treatment could not (Asd- derivatives). We observed by electrophoresis three plasmid bands in T. halophila D10; all Asd- derivatives lost the largest of these bands. This plasmid, pD1, has two SalI sites. We cloned and sequenced the 10 kb SalI fragment. The DNA sequence suggests that this fragment contains the aspartate decarboxylating trait.
A cellobiohydrolase I (cbhI) and a β-glucosidase 1 (bgl1) gene of Aspergillus aculeatus were expressed in Saccharomyces cerevisiae. The transformed cells secreted the enzymes efficiently in an active form. The recombinant CBHI gave two bands of different molecular mass (110 and 90 kDa) and the recombinant BGL1 gave one band (180 kDa) by SDS-PAGE. The recombinant CBHI and BGL1 had the same enzymatical properties as the native enzyme except for the specific activity toward cellulosic substrates. By the combination of three different types of cellulases, FI-CMCase, CBHI, and BGL1, we could hydrolyze Avicel up to 59% under our experimental conditions.