The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Selection ofRhizopus strains forl(+)-lactic acid andγ-linolenic acid production

The production ofl(+)-lactic acid and formation ofγ-linolenic acid by 50Rhizopus strains growing on saccharidic substrates were investigated. Formation of acids was observed on solid cultivation media but mainly during submerged fermentation. Strains with the highest selectivity of bothl(+)-lactic acid production andγ-linolenic acid formation were tested in a laboratory fermenter. The best producer was treated by UV irradiation to increase the fatty acid content in the biomass, especially that ofγ-linolenic acid. The conversion of 10% saccharidic substrate by this newly prepared strainRhizopus arrhizus CCM 8109 results in more than 95% of theoretical yield ofl(+)-lactic acid and permits a volume productivity of 0.4 gγ-linolenic acid per liter.     

Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid

Cucumber (Cucumis sativus L.) leaf explants were cultured either continuously on standard medium containing 4.5 µM 2,4- dichlorophenoxyacetic acid (2,4-d) and 4.4 µM benzylaminopurine, or first cultured for various periods at different levels of 2,4-d, picloram or naphthaleneacetic acid (NAA), and then transferred to standard medium. When cultured continuously on standard medium, less than 10% of the explants formed embryogenic callus. Initial culture on picloram or NAA, or on 2,4-d at a low concentration (1.4 µM) did not result in any embryogenic callus formation. Embryogenic callus formation increased to 40% if during the initial phase of the culture (10 days), the 2,4-d concentration was raised to 14 µM. Prolonged culture on 14 µM 2,4-d resulted in less embryogenic callus formation.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Aerobic electron transport in Propionibacterium shermanii effects of cyanide

Cyanide inhibited d- and l-lactate and NADH oxidase activities of membrane particles from Propionibacterium shermanii but only at relatively high concentrations. Inhibition occurred at two different sites in the electron transport pathway. One site, with a half-maximal inhibition concentration (I _0.5) of 2 to 3 mM KCN, is located at the terminal oxidase involved in cytochrome b oxidation; the evidence is consistent with cytochrome d being the major oxidase involved. At high concentrations, cyanide inhibited reduction of cytochrome b by d-lactate (I _0.5 value 20–25 mM cyanide). A proportion of the oxygen-uptake remained uninhibited even by 100 mM cyanide; this proportion was about 80% for succinate, 30% for l-lactate, 15% for d-lactate and 10% for NADH. The oxygen uptake per mol of substrate oxidised increased with increasing cyanide concentration and was accompanied by the formation of hydrogen peroxide as a product of a cyanide-insensitive oxidase system.Abbreviations PMS Phenazine methosulphate     

Long‐Chain O‐Ascarosyl‐alkanediols Are Constitutive Components of Caenorhabditis elegans but Do Not Induce Dauer Larva Formation

Two long‐chain ascarosides, O‐ascarosylnonacosane‐2,28‐diol ( 1 ) and O‐ascarosyluntriacontane‐2,30‐diol ( 2 ), were isolated from Caenorhabditis elegans and detected in all developmental stages of the worm. The long‐chain ascarosides were shown to be minor lipid components, and it was also shown that they do not induce dauer larva formation.     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

Depression of by-product formation during l-isoleucine production by a living-cell reaction process

Summary Two unnatural and unwanted amino acids, norvaline (Nva) and O-ethylhomoserine (O-EH) are formed as by-products in l-isoleucine production by Brevibacterium flavum AB-07 using a new process named the living cell reaction process. Nva formation was depressed by using a leucine auxotrophic mutant (AB-07-Leu-2) derived from strain AB-07. It was found that Nva formation was closely related to leucine biosynthesis. O-EH formation was repressed by addition of l-methionine to the reaction mixture. However, the homoserine-O-acetyltransferase of AB-07-Leu-2 was not subject to either inhibition or repression by addition of l-methionine. Furthermore, the O-EH-forming enzyme, which converts O-acetylhomoserine to O-EH, was speculated to be repressed by l-methionine. Offprint requests to: H. Yukawa     

Numerical taxonomy ofKlebsiella pneumoniae strains isolated from clinical and nonclinical sources

A total of 180 clinical and nonclinical isolates ofKlebsiella pneumoniae, for which 99 characteristics were recorded, were subjected to numerical taxonomy analysis. Of these strains, 172 clustered into five major groups, with an overall similarity of 64%. Intragroup similarities ranged from 77 to 82%, with the subgroups corresponding to the speciesK. pneumoniae sensu stricto, K. oxytoca, andKlebsiella spp. 1, 2, and 3. Biochemical tests useful in distinguishing the species included production of indole, degradation of pectate, growth at 10°C, fecal coliform response, production of urease, fermentation of inulin andd-tartrate, utilization ofl-arginine and gentisate andm-hydroxybenzoate, and pigment formation ond-gluconate ferric citrate agar.     

Gum Formation by Methyl Jasmonate in Tulip Shoots is Stimulated by Ethylene

The promotive effect of methyl jasmonate (JA-Me) on the induction of gum in tulip shoots (Tulipa gesneriana L. cvs. Gudoshnik and Apeldoorn) was studied in the presence of ethylene. Gum formation in the stem and the basal part of the leaves was induced by JA-Me (1% w/w in lanolin) and stimulated strongly by the simultaneous application of 1 or 5 mm 1-aminocyclopropane-1-carboxylic acid (ACC). JA-Me at a concentration of 0.1% did not induce gum, but that together with ACC at a concentration of 1 or 5 mm induced it substantially. Although JA-Me stimulated ethylene production substantially in the stem of intact tulips, ethephon (1% w/w) or ACC (1 or 5 mm) did not induce gum formation in tulip shoots. JA-Me induced gum formation in tulip shoots even in the presence of aminooxyacetic acid or cobalt ions. Moreover, gum formation was also observed in the cut shoot applied with JA-Me as a solution at concentrations of 0.23 mm or more. These results strongly suggest that JA-Me is required for gum formation in tulip shoots, and ethylene probably makes the tissues of shoots sensitive to JA-Me. Received March 23, 1998; accepted June 10, 1998     

Aerobic dissimilation of d-ribulose by yeasts

Aerobic dissimilation ofd-ribulose by various genera and species of yeasts was examined. Most strains tested utilizedd-ribulose fairly well, andd-arabitol was accumulated in the fermented broth but ribitol was not found. Torulopsis famata ATCC 20214,T.mannitofaciens CBS 5981 andT.versatilis CBS 1752 producedd-arabitol with a yield of 26 to 67% of the sugar used. Extracellular and intracellular formation of pentitol fromd-ribulose were compared with those fromd-xylulose.We wish to express our sincerest thanks to Prof. K. Arima and Prof. Y. Ikeda of the University of Tokyo for their kind guidance. We also wish to thank Dr. M. Mogi and Dr. N. Iguchi of this Institute for their encouragement and Mr. K. Kouchi for his technical assistance.     

Cell wall composition and synthesis via Golgi-directed scale formation in the marine eucaryote,Schizochytrium aggregatum,with a note on Thraustochytrium sp.

Summary Cell walls of Schizochytrium aggregatum and Thraustochytrium sp. were mechanically isolated and subjected to chemical analysis. On a dry weight basis the cell walls contain 21–36% carbohydrate and 30–43% protein. The principal sugar (>95%) of the Schizochytrium wall is l-galactose, while the Thraustochytrium cell wall contains l-galactose, d-galactose and xylose with l-galactose predominating. Ultrastructurally the cell walls of both organisms consist of a laminated structure which yields thin, flexible, nearly circular scales (0.5–1.1 in diameter) upon sonic disintegration. Structures presumed to be developing wall scales are found within cisternae of the Golgi apparatus in both organisms. The chemical composition and method of formation of the cell wall in these two protists is distinctly different from that found in the Saprolegniales (Oomycetes), the group with which these organisms have hitherto been aligned.     

d-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli

Recently, we reported on the construction of a whole-cell biotransformation system in Escherichia coli for the production of d-mannitol from d-fructose (Kaup B, Bringer-Meyer S, Sahm H (2004) Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation. Appl Microbiol Biotechnol 64:333–339). Supplementation of this strain with extracellular glucose isomerase resulted in the formation of 800 mM d-mannitol from 1,000 mM d-glucose. Co-expression of the xylA gene of E. coli in the biotransformation strain resulted in a d-mannitol concentration of 420 mM from 1,000 mM d-glucose. This is the first example of conversion of d-glucose to d-mannitol with direct coupling of a glucose isomerase to the biotransformation system.     

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH₂ and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.     

Inhibition of sulfate-forming activity in rat liver mitochondria by (aminooxy)acetate

Summary The effect of (aminooxy)acetate, an inhibitor of aminotransferases, on the sulfate formation froml-cysteine andl-cysteinesulfinate in rat liver mitochondria was studied. Incubation of 10 mMl-cysteine with rat liver mitochondria at 37°C in the presence of 10 mM 2-oxoglutarate and 10 mM glutathione resulted in the formation of 4.60 and 1.52µmol of sulfate and thiosulfate, respectively, per 60 min per mitochondria obtained from 1 g of liver. Under the same conditions sulfate formation froml-cysteinesulfinate was 24.96µmol, but thiosulfate was not formed. The addition of (aminooxy)acetate at 2 mM or more completely inhibited the sulfate and thiosulfate formation froml-cysteine and the sulfate formation froml-cysteinesulfinate. These findings support our previous conclusion that cysteine transamination and 3-mercaptopyruvate pathway (MP pathway) are involved in the sulfate formation froml-cysteine in rat liver mitochondria (Ubuka et al., 1992).     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Seasonal variants of anabaenopsis Raciborskii Wolosz

Summary Anabaenopsis Raciborskii Wolosz. has been described in detail with special reference to its morphological variants, which sometimes appear similar to Raphidiopsis indica Singh and Cylindrospermum species. The variants have been shown to be seasonal in distribution. Details of heterocyst and akinete formation have been followed.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Metabolism of d(+)-carnitine by Escherichia coli

Summary Escherichia coli 044 K74 grown under anaerobic conditions in the presence of l(–)-carnitine is able to convert d(+)-carnitine into the l(–)-enantiomer. This activity is repressed by electron acceptors such as oxygen and nitrate as well as by glucose. d(+)-Carnitine shows no effect on the induction or repression of the corresponding enzyme or enzyme system. Resting cells of E. coli 044 K74 were used for the formation of l(–)-carnitine from d(+)-carnitine. The maximum obtained yield was 50%. -Butyrobetaine was formed as a by-product. Offprint requests to: H. Jung     

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO₂, 0.05% pCO₂) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.     

Effect of environmental conditions on sclerotia and cleistothecia production in aspergillus

Summary Literature pertaining to sclerotial Aspergilli has been reviewed in brief. Observations on the effect of certain environmental conditions viz. pH, light, temperature of incubation, oxygen-deficient conditions and various relative humidity values on sclerotia production byAspergillus niger van Tieghem, (two strains),A. flavus Link (two strains),A. sclerotiorum Hüber (one strain) andA. paradoxus Fennell &Raper (one strain) and on cleistothecia production byA. nidulans (Eidam)Wint. (one strain) have been presented. Optimum pH for sclerotia or cleistothecia production was 7.5. In other respects sclerotia and cleistothecia behaved similarly. In general, condition showing maximum sclerotia or cleistothecia production was the one that showed maximum vegetative growth. Certain strains of the same species reponded differently to the same condition. Light completely inhibited sclerotia formation in one strain ofA. flavus. InA. paradoxus, in general, conditions favouring sclerotia production were those that inhibited (or retarded) the formation of conidial heads and the yellow pigment in the medium. Oxygen-deficient conditions inhibited or retarded sclerotia or cleistothecia formation. Production of sclerotia and cleistothecia increased with an increase in relative humidity values. No definite correlation could be observed between extent of sporulation and sclerotia or cleistothecia production except in case of relative humidity. Parallelism in the behaviour of sclerotia and cleistothecia production inAspergillus lends further support in favour of the hypothesis that in this genus sclerotia are sterile stromata.