Effect of organic cosolvent on kinetic resolution of tert-leucine by penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-dl-tert-leucine (N-Phac-dl-Tle) to produce l-tert-leucine (l-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-dl-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than −0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (ε) of organic cosolvents. The yields of l-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

On the interplay of environmental factors affecting taxis and motility in Rhodospirillum rubrum

Summary Resting suspensions of Rhodospirillum rubrum exhibit a positive chemotaxis toward sulfhydryl compounds, ATP, ADP, and oxidizable organic substrates such as malate and yeast extract.A negative chemotaxis is induced by various oxidizing substances, extremes of p _h , and poisons (especially thiol inhibitors).Positive aerotaxis, displayed in the dark, is inhibited by reducing substances and reversed by strong illumination.Positive aerotaxis and phototaxis are augmented by organic compounds which serve as metabolic substrates; they are suppressed by ADP and ATP. Positive aerotaxis is suppressed by moderate illumination; phototaxis is suppressed by air.These effects are discussed in terms of the hypothesis of Links that tactic responses are associated with a decrease in the energy available to the locomotor apparatus. A correlation of taxis with the level of oxidation of the electron transport system (cytochromes, etc.) fails. The possibility is discussed that taxis and motility are governed by a central, nerve-like coordinating system. An hypothesis similar to that of Links but embracing the possibility of central coordination of taxis is offered.     

Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

Production of d-amino acids using immobilized d-hydantoinase from lentil, Lens esculenta, seeds

d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries. Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998     

Construction and characterization of a recombinant whole-cell biocatalyst of Escherichia coli expressing styrene monooxygenase under the control of arabinose promoter

A recombinant Escherichia coli (pBAB1) containing styrene monooxygenase (SMO) was developed for the conversion of styrene to enantiopure (S)-styrene oxide that is an important chiral building block in organic synthesis. The styAB genes encoding SMO was cloned into a multicopy plasmid under the tightly regulated promoter of bacterial l-arabinose operon which is inducible by l-arabinose. The recombinant showed that expression level of StyA protein and whole-cell SMO activities were varied depending on the concentration of the inducer l-arabinose. The maximum SMO activity was 108 U/g cdw when the cells were induced with 0.2% l-arabinose. SDS-PAGE and Western blot analyses indicated that whole-cell SMO activity was strongly correlated with the expression level of StyA protein. Organic-aqueous two-phase experiment could yield 50 mM enantiopure (S)-styrene oxide in organic phase in 18 h, but the recombinant SMO activity was unstable during the reaction. The expression of styAB under the control of l-arabinose promoter was significantly repressed in the presence of glucose.     

Some effects of organic acid supplementation on utilization of ammonium compounds by three pathogenic fungi

Utilization of ammonium nitrogen byFusarium moniliforme Sheld.,Curvularia verruciformis Agarwal &Sahni andSclerotium rolfsii Sacc., with and without organic acid supplementation was studied. Ammonium utilization by these organisms generally improved with small amounts of organic acid supplementation except in the case ofC. verruciformis on ammonium chloride where fumaric acid was observed to suppress utilization. Effects of supplementation were seen to be dependent on the nature of the organic acid, the ammonium source used and the organism employed. Further evidence obtained by varying the quantities of succinic acid in ammonium chloride media showed that higher levels of supplementation gave increased growth of the fungi and prevented the pH from falling. It seems that these acids play a dual role as pH buffers as well as nutritional compounds.     

Enantioselective synthesis ofN-acetyl-l-methionine with aminoacylase in organic solvent

We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl₂, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids.     

Utilization of organic nitrogen compounds by yeasts of the genusSaccharomyces

Yeasts belonging to 27 species ofSaccharomyces were tested for their ability to used-amino acids,l-lysine and various amines and amides as nitrogen source. Most yeasts capable of growing onl-lysine or amines could utilized-amino acids.Saccharomyces (sensu strictu) have a very limited ability to grow on the organic nitrogen compounds tested. However, there is no obvious relationship between the utilization of these compounds and the proposed divisions of the genusSaccharomyces.Issued as N.R.C. No. 9845.     

Extensions to Kiritani and Nakasuji’s method for analysing insect stage-frequency data

Summary There is evidence that the simple method ofKiritani andNakasuji (1967) for the analysis of insect stage-frequency data produces estimates of stage-specific survival rates that compare well with the estimates from more complicated methods (Manly, 1975). However the method as originally proposed byKiritani andNakasuji cannot always be applied because it assumes that the insect population involved was sampled at regular intervals of time. Furthermore, parameters such as the durations of stages are not estimated. In this note modifications to the basic method are suggested with the idea of overcoming these limitations.     

Nutritional and medicinal aspects of <Emphasis Type="SmallCaps">d</Emphasis>-amino acids

This paper reviews and interprets a method for determining the nutritional value of d-amino acids, d-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a nutritionally essential amino acid such as l-lysine (l-Lys), l-methionine (l-Met), l-phenylalanine (l-Phe), and l-tryptophan (l-Trp) as well as the semi-essential amino acids l-cysteine (l-Cys) and l-tyrosine (l-Tyr). The results show wide-ranging variations in the biological utilization of test substances. The method is generally applicable to the determination of the biological utilization and safety of any amino acid derivative as a potential nutritional source of the corresponding l-amino acid. Because the organism is forced to use the d-amino acid or amino acid derivative as the sole source of the essential or semi-essential amino acid being replaced, and because a free amino acid diet allows better control of composition, the use of all-amino-acid diets for such determinations may be preferable to protein-based diets. Also covered are brief summaries of the widely scattered literature on dietary and pharmacological aspects of 27 individual d-amino acids, d-peptides, and isomeric amino acid derivatives and suggested research needs in each of these areas. The described results provide a valuable record and resource for further progress on the multifaceted aspects of d-amino acids in food and biological samples.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

<Emphasis Type="SmallCaps">l</Emphasis>-Carnitine induces recovery of liver lipid metabolism in cancer cachexia

Cancer cachexia causes metabolic alterations with a marked effect on hepatic lipid metabolism. l-Carnitine modulates lipid metabolism and its supplementation has been proposed as a therapeutic strategy in many diseases. In the present study, the effects of l-carnitine supplementation on gene expression and on liver lipid metabolism-related proteins was investigated in cachectic tumour-bearing rats. Wistar rats were assigned to receive 1 g/kg of l-carnitine or saline. After 14 days, supplemented and control animals were assigned to a control (N), control supplemented with l-carnitine (CN), tumour-bearing Walker 256 carcinosarcoma (TB) and tumour-bearing supplemented with l-carnitine (CTB) group. The mRNA expression of carnitine palmitoyltransferase I and II (CPT I and II), microsomal triglyceride transfer protein (MTP), liver fatty acid-binding protein (L-FABP), fatty acid translocase (FAT/CD36), peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and organic cation transporter 2 (OCTN2) was assessed, and the maximal activity of CPT I and II in the liver measured, along with plasma and liver triacylglycerol content. The gene expression of MTP, and CPT I catalytic activity were reduced in TB, who also showed increased liver (150%) and plasma (3.3-fold) triacylglycerol content. l-Carnitine supplementation was able to restore these parameters back to control values (p < 0.05). These data show that l-carnitine preserves hepatic lipid metabolism in tumour-bearing animals, suggesting its supplementation to be of potential interest in cachexia.     

Occurrence and Bacterial Cycling of d Amino Acid Isomers in an Estuarine Environment

Abundance of d isomers of amino acids has been used in studies of organic matter diagenesis to determine the contribution of bacterial biomass to the organic matter, especially in marine sediments. However, fluxes of d amino acids in pelagic waters are poorly known. Here we present seasonal changes (March–September) in concentrations of dominant d amino acids in the pool of dissolved free and combined (hydrolysable) amino acids (DFAA and DCAA) in the shallow Roskilde Fjord, Denmark. The amino acid dynamics are related to pelagic bacterial density and activity and abundance of viruses. d␣isomers made up 3.6 and 7.9% of the DFAA and DCAA (average values), respectively, and had similar seasonal variations in concentrations. In batch cultures (0.7- and 0.2-m filtered water in a 1:9 mixture) microbial activity reduced l+d DCAA concentrations in seven of ten sampling dates, while DCAA were released at the remaining three sampling times. NH₄⁺ balance (uptake or release) in the cultures correlated significantly with variations in concentrations of d-DCAA, but not with the total DCAA pools. Abundance of viruses did not correlate with density or production of bacteria in the fjord, but covaried with mineralization of total C, DCAA and PO₄³⁻ in the batch cultures. The content of d amino acids in bacterial biomass in the cultures varied from 6.7 to 12.5% and correlated with the d isomer concentration in the fjord, except for d-Ala. In an additional six-day batch culture study, DCAA and d-DCAA were assimilated by the bacteria during the initial 36 h, but were released between 36 and 42 h simultaneous with a decline in the bacterial density. Our results demonstrate that peptidoglycan components contribute to natural amino acid pools and are assimilated by bacterial assemblages. This cell wall “cannibalism” ensures an efficient recycling of nutrients within the microbial community. Significant positive correlations between viral abundance and bacterial mineralization of organic matter in the fjord indicated that viral lysis contributed to this nutrient recycling.     

Inhibitory effects of organic sulfur compounds on Histoplasma capsulatum

Summary Eight classes of organic sulfur compounds, comprising 42 substances, were tested in vitro for activity against the yeast phase ofHistoplasma capsulatum Darling. Activity was found in the classes of thiols, thio acids, disulfides, and thiolsulfonates. The classes of sulfinates, sulfenamides, penicillamine analogs, and reaction products of thiols with aldehydes or ketones were unpromising. Tentative conclusions are drawn of structure-activity relations within classes as a guide to coupling of classes to form disulfides.This investigation was supported by Biomedical Science Support Grant, National Institutes of Health Grant FR-07089-02 to Vanderbilt University (Ilda McVeigh) and by PHS Research Grant AM-11685 from the National Institute of Arthritis and Metabolic Diseases (Lamar Field).     

Chum salmon trypsin-catalyzed preferential formation of peptides containing D-amino acid

Summary. Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from N ^α -(tert-butyloxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. All substrates tested in this study undergo less enantioselective coupling reaction, and the coupling product was the favorably obtained d-series rather than l-series (in the present case; N ^α -Boc-d-Ala and N ^α -Boc-l-Ala). The optimum condition for the coupling reaction was studied by changing the organic solvent, buffer solution, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible. Received August 10, 2000 Accepted December 2, 2000     

Ionic liquid catalyzed synthesis and characterization of heterocyclic and optically active poly (amide-imide)s incorporating <Emphasis Type="SmallCaps">l</Emphasis>-amino acids

N,N′-Pyromelliticdiimido-di-l-alanine (1), N,N′-Pyromelliticdiimido-di-l-phenylalanine (2), and N,N′-Pyromelliticdiimido-di-l-leucine (3) were prepared from the reaction of Pyromellitic dianhydride with corresponding l-amino acids in a mixture of glacial acetic acid and pyridine solution (3/2 ratio) under refluxing conditions. A series of poly (amide-imide)s containing l-amino acids were prepared from the synthesized dicarboxylic acids with two synthetic aromatic diamines in an ionic liquid (IL) as a green, safe and eco-friendly medium and also reactions catalysis agent. Evaluation of data shows that IL is the better polyamidation medium than the reported method and the catalysis stand on the higher inherent viscosities of the obtained PAIs and the rate of polymerizations beyond the greener reaction conditions and deletion of some essential reagents in conventional manners. Characterization were performs by means of IR, MS and ¹H NMR spectroscopy, elemental analysis, specific rotation, thermogravimetric analysis and differential scanning calorimetric techniques. Molecular weights of the obtained polymers were evaluated viscometrically, and the measured inherent viscosities were in the range 0.43–0.85 dL/g. These polymers were readily soluble in many organic solvents. These polymers still kept good thermal stability with glass transition temperatures in the range of 94–154°C, and the decomposition temperature under the nitrogen atmosphere for 10% weight-loss temperatures in excess of 308°C.     

Zur Kenntnis von Leucothrix,einer Gattung farbloser Cyanophyceen

Zusammenfassung Leucothrix ist eine marine, fädige, apochlorotische Cyanophycee. Wo Meeresalgen in Zersetzung übergehen, ist sie fast immer zu finden. Durch Ausstreichen auf Seewasseragar mit wenig Fleischextrakt können Reinkulturen hergestellt werden.Die meisten Stämme von verschiedenen Küsten sind einander morphologisch und physiologisch so ähnlich, daß sie zur selben Art Leucothrix mucor Oersted zu stellen sind, wenn auch unbedeutende Unterschiede bestehen.Ein Ammonsalz in Seewasser mit den üblichen Nährsalzen und einer Hexose oder Salzen einiger organischer Säuren ermöglicht gutes Wachstum. Vitamine sind nicht nötig.Die Zugehörigkeit zu den Cyanophyceen ist durch die Bildung von Hormogonien mit Gleitvermögen, die Beschaffenheit der Zellfäden und das Auftreten von Nekridien gesichert.

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Summary A colourless marine filamentous organism occurring frequently on decaying seaweeds has been repeatedly described under various names until Harold u. Stanier (1955) identified it with Leucothrix mucor (Oversted, 1844). They also demonstrated its morphological features which show that the organism belongs as an apochlorotic member to the Cyanophyceae.The present author, after adding a few morphological details, described a second species as Leucothrix cohaerens (Pringsheim, 1957). The strains derived from a number of coastal localities newly isolated for nutritional investigations or given to me by other authors, all belong to the original type. If there are differences they are very small.Clonal axenic strains were prepared by streaking on seawaterbeefextract agar plates. They were tested for their nutritional requirements in liquid media with artificial seawater, the customary nutrient salts and organic substrates. With an ammonium salt as nitrogen source glucose and other hexoses supported the growth of Leucothris mucor, various organic acids to a lesser degree. Vitamins are not required but substances like beefextract and yeast-autolysate make the growth richer.The taxonomic position of Leucothrix as related to the Cyanophyceae is established by the production of hormogonia with gliding motility, the structure of the trichomes, and the occurrence of necridia.