Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Metabolic design in the amino-acid-producing bacteriumCorynebacterium glutamicum

The Gram-positive bacteriumCorynebacterium glutamicum is used for the industrial production of amino acids,e.g. ofl-glutamate andl-lysine. By cloning and expressing the various genes of thel-lysine pathway inC. glutamicum we could demonstrate that an increase of the flux ofl-4-aspartaldehydate tol-lysine could be obtained in strains with increased dihydro-dipicolinate synthase activity. Recently we detected that inC. glutamicum two pathways exist for the synthesis ofdl-2,6-diaminopimelate andl-lysine. Mutants defective in one pathway are still able to synthesize enoughl-lysine for growth but thel-lysine secretion is reduced to 50–70%. Using NMR-spectroscopy we could calculate how much of thel-lysine secreted into the medium is synthesizedvia the one and the other pathway. Amplification of the feedback-inhibition-insensitive-homoserine dehydrogenase and homoserine kinase in a highl-lysine-overproducing strain made it possible to channell of the carbon flow from the intermediate 4-aspartaldehydate toward homoserine, resulting in a high accumulation ofl-threonine. For a further flux froml-threonine tol-isoleucine the allosteric control of threonine dehydratase was eliminated. Dedicated to Dr. Z. Vaněk on the occasion of his 70th birthday Presented at theIUMS Congresses '94-7th International Congress of Bacteriology and Applied Microbiology Division, Prague, July 3–8, 1994 (Bacteriological Symposium BS-12Regulation of Microbial Product Overproduction).     

Enzymic synthesis ofL-Lysine fromDL-α-Amino-ε-caprolactam by new microbial strains

The production of L-lysine fromDL-α-amino-ε-caprolactam (DL-ACL) by new strains producingL-α-amino-ε-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis ofL-ACL byCryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified asPseudomonas sp. were determined. Synthesis ofL-α-amino-ε-caprolactamase is induced byDL-ACL orL-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10 % aqueousDL-ACL toL-lysine by a mixture of native cells in a mass ratio of 1: 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 °C and pH 8.0     

Influence of the ion-composition of the medium on alkaloid production by “hairy roots” ofDatura stramonium

The influence of culture age on biomass production and alkaloid yield of “hairy roots” obtained after infection ofDatura stramonium L. withAgrobacterium ATCC 15834 was investigated. Maximal hyoscyamine yield was obtained with roots harvested after six weeks. Fluctuations were found for tropine yield, the precursor of the ring moiety of hyoscyamine. These indicate a continuous conversion to hyoscyamine during the exponential growth phase. The effect of the ion-balance was investigated by preparing five different media that only differed in their ionic composition. The ionic interactions between macroelements, differently influenced biomass production and alkaloid yield. As a result, highest biomass yield was found with NO₃ ⁻ - and K⁺-dominance, whereas hyoscyamine yield was highest with the culture medium in which SO₄ ²⁻ and K⁺ were dominant. Shifting the intercationic balance to strongly towards Ca²⁺ caused an overall reduced metabolism, since as well biomass yield as hyoscyamine yield was lowest with the NO₃ ⁻Ca²⁺-medium. Also tropine yield was affected by the ion-balance, indicating that this culture parameter also influences alkaloid synthesis.[/p]     

Different affinities of the α- and β-anomers ofD-glucose,D-mannose andD-xylose for the glucose uptake system of baker’s yeast

A recording technique for measuring the sugar uptake by cell suspensions using a polarimeter is described. The method makes it possible to calculate the uptake rates of the α-and β-anomers. The constitutive monosaccharide transport system ofSaccharomyces cerevisiae andSaccharomyces fragilis exhibits a higher affinity for the α-anomers ofd-glucose,d-manose andd-xylose than for the corresponding β-anomers, this resulting in a preferential uptake of the α-anomers from a mixture. The α-anomer ofd-xylose is preferred both during influx and efflux. The membrane transport ofd-xylose inSaccharomyces cerevisiae is not associated with a change of the anomer configuration. The facilitated diffusion system appears to possess a regulatory role for the utilization ofd-glucose andd-mannose in both yeast species investigated.     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Morphological and cytological investigations ofUlothrix zonata and taxonomy of the related species

The present communication deals with the observations made on the morphology, reproduction and cytology ofUlothrix zonata (Weber & Mohr)Kütz. in culture. The alga displays a remarkable phenotypic plasticity in nature as well as in culture. The present study provides additional evidence of karyology in support ofLokhorst andVroman's treatment ofU. zonata which merges a number of earlier described species in it. The cytological details and chromosome number (n = 10) determined for the Indian isolates ofU. zonata agree with those ofSarma for the British material.NBRI Research Publication No. 63 (N.S.)     

Asymmetric synthesis of unnaturall-amino acids using thermophilic aromaticl-amino acid transaminase

Aromaticl-amino acid transaminase is an enzyme that is able to transfer the amino group froml-glutamate to unnatural aromatic α-keto acids to generate α-ketoglutarate and unnatural aromaticl-amino acids, respectively. Enrichment culture was used to isolate thermophilicBacillus sp. T30 expressing this enzyme for use in the synthesis of unnaturall-amino acids. The asymmetric syntheses ofl-homophenylalanine andl-phenylglycine resulted in conversion yields of >95% and >93% from 150 mM 2-oxo-4-phenylbutyrate and phenylglyoxylate, respectively, usingl-glutamate as an amino donor at 60°C. Synthesizedl-homophenylalanine andl-phenylglycine were optically pure (>99% enantiomeric excess) and continuously pre-cipitated in the reaction solution due to their low solubility at the given reaction pH. While the solubility of the α-keto acid substrates is dependent on temperature, the solubility of the unnaturall-amino acid products is dependent on the reaction pH. As the solubility difference between substrate and product at the given reaction pH is therefore larger at higher temperature, the thermophilic transaminase was successfully used to shift the reaction equilibrium toward rapid product formation.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

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.     

Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.     

Synthesis and photophysical properties of L-N ε-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-lysine,dabcyl-like chromophore for peptide studies

Summary A synthesis ofl-N^ɛ-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-lysine [Lys(AQN)], the dabcyl-like chromophore, and its derivatives useful in peptide chemistry is described. N^α-tert-butoxycarbonyl derivative of the title compound was obtained in a good yield using aromatic nucleophilic substitution reaction. In this form, or after conversion to the N^α-fluorenylmethoxycarbonyl derivative, it can be directly used in the solid phase peptide synthesis using either Boc- or Fmoc-strategy.     

Evidence for two β-galactosidase activities inStreptomyces violaceus

The kinetics of β-galactosidase synthesis induced by galactose and lactose inStreptomyces violaceus, as well as the pattern ofo-nitrophenyl-β-d-galactoside-positive bands observed after electrophoresis of both crude extracts, showed the presence of different β-galactosidase activities in the two cellular extracts. It is postulated that the lactase activity induced by lactose is the physiological enzyme responsible for lactose utilization. The possible function of the galactose-induced activity is also discussed.     

Utilization of oligosaccharides by three imperfect fungi

Summary The utilization of oligosaccharides (maltose, sucrose, lactose and raffinose) by three imperfect fungi viz.Alternaria tenuis Auct.,Colletotrichum capsici (Syd.)Butler &Bisby andColletotrichum gloeosporioides Penz. isolated from diseased leaves of tomato,Scindapsus pictus andPolyscias balfuriana respectively, was studied chromatographically. Except for lactose, all the sugars were utilizzed through a hydrolytic pathway and almost all proved to be a suitable source of carbon for the growth of fungi. Along with the utilization of sucrose, a simultaneous synthesis of an oligosaccharide was also observed in case of two species ofColletotrichum. C. gloeosporioides, however, also synthesized an oligosaccharide, maltotriose if it is grown in the medium containing maltose. During the utilization of raffinose, melibiose and fructose were detectted in the culture medium ofA. tenuis. Melibiose was further broken down into glucose and galactose by the two species ofColletotrichum.All the sugars were consumed from the media within 15 days except for lactose which persisted in the media even after 15 days.     

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-dl-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, todl-5-fluorotryptophan anddl-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-dl-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,dl-p-fluorophenylalanine,dl-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-dl-aspartic acid, α-methyl-dl-methionine,N-o-nitrophenylsulfenyl-l-tryptophan, norleucine, andS-2-aminoethyl-l-cysteine did not show any significant effect. Treatment with 7-azatryptophan,dl-β-hydroxynorvaline,l-methionine-dl-sulfoximine,l-methionine sulfone, and β-2-thienyl-dl-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Summary Total retro-inverso (TRI) analogues of bradykinin (BK), the B_2a -selective kinin antagonistd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹, Val⁵, Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B_2a bradykinin receptor with affinities (K _d ) ranging from 64 μM to 4 μM. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than ‘agonists’. Three TRI peptidomimetics ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway. Received: 26 August 1998 / Accepted: 19 October 1998     

D-Amino acids of the amino acid pool and occurrence of racemase andD-amino acid oxidase activities inEscherichia coli B

Less than 20 % of the amino acid content of the amino acid pool ofEscherichia coli B exists in theD-form. Alanine, glutamic acid, and valine were shown by gas-chromatography to be partially in theD-form. OnlyD-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles butD-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid -D-amino acid - oxo acid which exists in the oxidation ofL-alanine does not exist with otherL-amino acids. It is likely that otherD-amino acids in the pool are formed in the presence ofD-amino acid oxidase orD-amino acid aminotransferase.