Improvement of L-Lactate Production by CYB2 Gene Disruption in a Recombinant Saccharomyces cerevisiae Strain under Low pH Condition

Using a DNA microarray, we found that expression of the genes related to lactate metabolism was upregulated in a lactate-producing recombinant Saccharomyces cerevisiae strain. Disruption of the CYB2 gene encoding L-lactate dehydrogenase improved the L-lactate production by S. cerevisiae under low pH condition.     

Effects of cis-Platin on t-RNA of Saccharomyces cerevisiae

We synthesized three hydantocidin derivatives and evaluated their herbicidal activity in order to elucidate the role of the spirohydantoin system at the anomeric center of hydantocidin. With application to foliage at 1000 ppm, only α-ureidoamide 14 demonstrated activity, the remaining compounds being found to be inactive.     

The Fermentative Formation of CDP-Choline by Haploid Cells of Yeasts and the Change of the Temperature-sensitivity of a Mutant of a Yeast,Saccharomyces rouxii

Phosphorylation of CMP and formation of CDP-choline were tested with various haploid cells of yeasts. Most of them had more or less the ability, but a mutant (Lys–M7, alpha type) of Saccharomyces rouxii was found to lack the ability. Further study revealed the change of the temperature-sensitivity of the mutant, which could not produce CDP-choline when treated at 37°C, whereas it could at 16°C. The growth of the mutant was more sensitive to temperatures than that of the wild strain. The former did not grow at 36.3°C, while the latter grew.     

NADP+-Dependent D-Arabinose Dehydrogenase Shows a Limited Contribution to Erythroascorbic Acid Biosynthesis and Oxidative Stress Resistance in Saccharomyces cerevisiae

The molecular aspects and physiological significance of NADP⁺-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP⁺-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H₂O₂ and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H₂O₂ with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.     

Purification of an Aspergillus oryzae Metallo-proteinase by Talopeptin-aminohexyl-Sepharose and Its Properties

Simple and speedy purification of Aspergillus oryzae metallo-proteinase was performed using Talopeptin-aminohexyl-Sepharose The properties of the metallo-proteinase were: optimum pH 6.5; pH stability, pH 5~11; optimum temperature,50°C; and molecular weight 42,000 (SDS electrophoresis). These results were similar to those of neutral protease I from Aspergillus oryzae reported by Nakadai et al. This metallo-proteinase was compared with others from microbes using the metallo-proteinase inhibitors FMPI, PLT, and Talopeptin. The metallo-proteinase is unique in the point at which FMPI and PLT gave nearly stoichiometrical inhibition.     

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.     

Novel Characteristics of Selenomonas ruminantium Lysine Decarboxylase Capable of Decarboxylating Both L-Lysine and L-Ornithine

Lysine decarboxylase (LDC; EC 4.1.1.18) of Selenomonas ruminantium is a constitutive enzyme and is involved in the synthesis of cadaverine, which is an essential constituent of the peptidoglycan for normal cell growth. We purified the S. ruminantium LDC by an improved method including hydrophobic chromatography and studied the fine characteristics of the enzyme. Kinetic study of LDC showed that S. ruminanitum LDC decarboxylated both L-lysine and L-ornithine with similar K _m and the decarboxylase activities towards both substrates were competitively and irreversibly inhibited by DL-α-difluoromethylornithine, which is a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17). We also showed a drastic descent of LDC activity owing to the degradation of LDC at entry into the stationary phase of cell growth.     

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.     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

Properties of a sucrose-tolerant Mutant of Saccharomyces cerevisiae

We characterized a sucrose-tolerant mutant of Saccharomyces cerevisiae, S22, that produces about four times as much acetate as the wild-type strain K9. We monitored the concentration of extracellular acetate during cultivation, and compared the gene expression ratios of S22 with those of K9 using DNA microarray. We propose that the sucrose tolerance of S22 may be related to the overexpression of the ENA1, ENA2, and ENA5 genes and some cell wall mannoprotein genes, and that the high acetate productivity of S22 is related to the overexpression of the ALD4 gene and oxidative phosphorylation genes.     

Isolation and Genetic Analysis of a Chloramphenicol-resistant Mutant of Pleurotus ostreatus

A chloramphenicol-resistant mutant was isolated by mutagenesis with manganese to make a study of the genetics and function of mitochondrial genes in the higher basidiomycete, Pleurotus ostreatus. The resistant mutant obtained was shown to grow well on media containing up to 4mg/ml chloramphenicol. The result of genetic analysis suggested that the origin of the factor for chloramphenicol resistance of the mutant might be mitochondrial.     

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.     

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.     

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.     

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 and Characterization,of Rice Bran Lipase II

A species of rice bran lipase (lipase II) was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose, Sephadex G–75 and CH-Sephadex C–50. Both polyacrylamide disc electrophoresis and ultracentrifugation demonstrated that the enzyme protein is homogeneous. The isoelectric point of the enzyme was 9.10 by ampholine electrophoresis. The sedimentation coefficient of the enzyme was evaluated to be 2.60 S, and the molecular weight to be 33,300 according to Archbald’s method. The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 27°C. It was stable over the pH range from 5 to 9.5 and below 30°C. In substrate specificity, the enzyme exhibited a high specificity toward triglycerides having short-carbon chain fatty acids, although it was capable of hydrolyzing the ester bonds in the rice and olive oil.     

Effect of Dietary l-Glutamine on the Hepatotoxic Action of d-Galactosamine in Rats

The protective effect of dietary l-glutamine against the hepatotoxic action of d-galactosamine (GalN) was investigated by model experiments with rats. Rats fed with 20% casein diets containing 10% free amino acids were injected with GalN, and the serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activities and the hepatic glycogen content were assayed 20 hours after the injection. These enzyme activities in the group fed with the 10% l-glutamine diet for 8 days were lower than those in the groups fed with the control, 10% l-glutamic acid and 10% l-alanine diets for 8 days. The more prolonged the feeding period with the 10% l-glutamine diet was, the more the serum activity levels of such enzymes were decreased. Although neomycin also lowered these enzyme activities, its simultaneous ingestion with neomycin did not show any additive or synergistic effect. The hepatic glycogen content in the 10% glutamine group still remained high after the GalN treatment. It is therefore assumed that the effectiveness of glutamine intake would have been mediated by glycogen metabolism rather than by uridine metabolism.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Isolation of a New Root Growth-stimulating Substance from a Fungus

In the last few decades, enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using tyrosine phenol-lyase (Tpl) has been industrialized. This method has an intrinsic problem of tyrosine contamination because Tpl is synthesized under tyrosine-induced conditions. Herein, we constructed a hyper-L-dopa-producing strain by exploiting a mutant TyrR, an activator of tpl. The highest productivity was obtained for the strain grown under non-induced conditions. It was 30-fold higher than that obtained for tyrosine-induced wild-type cells.