Stimulation of tylosin productivity resulting from cyclic feeding profiles in fed batch cultures

Summary Batch fermentations for the production of the macrolide antiobiotic tylosin were carried out with a medium in which the maximum specific rate of tylosin synthesis (q_tylosin) occurred while the producer organism Streptomyces fradiae, was actively growing. With this medium, the value of q_tylosin decreased rapidly throughout the fermentation. By using a microprocessor to control the feeding of monosodium glutamate, and glucose in cyclic square wave profiles, it was possible to minimize the decrease in q_tylosin observed in the control and to maintain a linear volumetric accretion rate of tylosin resulting in a 114% increase over control in the 250 hour tylosin titre.     

Production of the macrolide antibiotic tylosin in batch and chemostat cultures

The production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase–idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (q_tylosin) was found to be a function of the growth rate. The maximum value of (q_tylosin) was observed when D = 0.017 hr^?1. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr^?1, the maximum value of q_tylosin was 0.71 mg tylosin/g dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW-hr. This value of q_tylosin was 40% greater than the maximum q_tylosin observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of q_tylosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of q_tylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased the uptake of phosphate decreased the values of q_tylosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of q_tylosin of 1.1 mg/g/hr being obtained at a growth rate of 0.03 hr^?1. Batch fermentations with this medium did not follow a trophophase–idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.     

Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts

Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS absorption unit full scale - CHI chalcone isomerase (EC 5.5.1.6) - CHS chalcone synthase (EC 2.3.1.74) - C40H cinnamic acid 4-hydroxylase (EC 1.14.13.11) - CLE elicitor from Colletotrichum lindemuthianum - IFOH isoflavone 2-hydroxylase - IFS isoflavone synthase - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Symbiotic growth of a yeast and a bacteria in batch fermentors

Summary Single cell protein was produced from cassava starch by symbiotic growth of the -amylase producing bacteria Bacillus subtilis and the yeast Candida utilis, which is accepted as fodder. By batch fermentations it was shown, that the pH fluctuation during the fermentation and the inoculum were extremely important parameters.     

Catalysis of enzyme aggregates in organic solvents. An attempt at evaluation of intrinsic activity of proteases in ethanol

Summary Catalytic activities of -chymotrypsin and subtilisin Carlsberg for transesterification of N-acetyl-L-tyrosine methyl ester in ethanol markedly increased by decreasing the amount of the enzymes in the reaction mixtures. The results were kinetically attributed to changes in K_M and k_cat due to formation of smaller enzyme aggregates.     

Production of the macrolide antibiotic tylosin in cyclic fed-batch culture

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.     

Resolution of the extracellular α-glucosidase system ofBacillus sp. NCIB 11203

Summary Two extracellular -glucosidases (EC 3.2.1.20, -D-glucoside glucohydrolase) of the alkalophilic bacterium,Bacillus sp. NCIB 11203, were separated, purified and partially characterised. Resolution of the system into two separate enzymes was achieved by fractionation with (NH₄)₂SO₄ and chromatography on DEAE-Biogel A. The first of these activities, an -glucosidase, hydrolysed p-nitrophenyl--D-glucopyranoside preferentially and had minor activity on isomaltose and isomaltotriose. The second enzyme was a maltase and displayed highest activity on maltose and maltotriose and some activity on p-nitrophenyl--D-glucopyranoside.     

Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin

The effect of ammonium ions on growth and tylosin biosynthesis in Streptomyces fradiae NRRL 2702 cultured on a chemically defined medium was studied. Mycelial growth and tylosin production were not affected when ammonium sulphate was added to idiophase cultures to a final concentration of 10 mm or 20 mm; however, when ammonium sulphate was added to tylosin cultures to a final concentration of 20 mm before the onset of antibiotic biosynthesis (trophophase), tylosin production was severely suppressed while mycelial growth was stimulated. The activities of propionyl-coenzyme A carboxylase (EC 6.4.1.3) and methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1), enzymes involved in the synthesis of tylonolide precursors, were depressed in high ammonium cultures. The activity of macrocin 3′-o-methyltransferase, which catalyses the methylation of macrocin to form tylosin, was also affected by high concentrations of ammonium ions added in the trophophase.     

Cyclic AMP Diphenyl Phosphoric Mixed Anhydride: Synthesis,P NMR Characterization and Reaction with Dimethylamine

Abstract

Phosphorus diastereoisomers, R _p and S _p of p¹-adenosine cyclic 3′, 5′ P² -diphenylpyrophosphate (cyclic AMP diphenylphosphoric mixed anhydride) (1) were prepared from adenosine cyclic 3′, 5′-monophosphate (cyclic AMP) and diphenyl phosphorochloridate and characterized by ³¹p NMR. The synthesis preferentially gave R _p-1. Reaction of 1 with dimethylamine resulted in the formation of a (～ 3:1) mixture of adenosine cyclic 3′,5′-N, N-dimethylphosphoramidate and diphenyl-N, N-dimethylphosphoramidate and occurred with inversion of configuration at cyclic AMP phosphorus.     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Continuous-culture studies on the regulation of tylosin biosynthesis

The metabolic regulation of tylosin synthesis by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture a medium which diminished the trophophase-idiophase kinetic pattern was used to assess the activities of the enzymes involved in tylosin synthesis. The enzymes methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) showed early enzymatic derepression, both enzymes reaching their highest specific activities after 72-96 fermentation. The activity of macrocin 3' -O-methyltransferase, the enzyme catalyzing the conversion of macrocin (tylosin C) to tylosin (tylosin A). also peaked at 72 h. The specific activities of the three enzymes showed close correlation with the q(tylosin) value. In chemostat cultures the activities of the enzymes and the intracellular level of the adenylate pool and energy charge were studied as a function of dilution rate. Under steady-state conditions, increases in the specific growth rate repressed the enzymes activities with a concomitant increase in the intracellular level of the adenylate pool, while the adenylate energy charge remained almost constant and in the range 0.5-0.52. The highest specific activities of the enzymes were observed when D = 0.008 h (-1). The specific rate of tylosin synthesis was inversely proportional to the specific growth rate and the intracellular level of adenylate pool. The pool of adenylate could be a nutritional parameter which had a considerable influence on the biosynthesis of tylosin.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

The effect of temperature on the kinetics of ethanol production by strains of Zymomonas mobilis

Summary Two strains of Z. mobilis were evaluated for temperature sensitivity between 25°C and 40°C. At higher temperatures the cell viability, biomass yield, ethanol yield and final ethanol concentration decreased, and there was evidence of increased ethanol inhibition. However the kinetic parameters , q_s and q_p were largely unaffected by temperature over this range.     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Some characteristics of the cellulolytic enzyme system of Chaetomium cellulolyticum

Summary The effects of pH and temperature on the activities of endoglucanase, exoglucanase and -glucosidase of C. cellulolyticum were studied. Thermal stability of these enzymes was characterized. Enzymatic hydrolyses of cellulose were performed yielding predominantly glucose and cellobiose. Glucose was shown to be a potent inhibitor of its own formation in cellulose saccharification.     

DNA-[N4-Cytosine]-Methyltransferase from Bacillus amyloliquefaciens: Mechanism of Action Derived from Steady-State Kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5"-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the K _M values (0.107 M for DNA and 1.45 M for SAM), which means that no nonreactive enzyme–substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAMDNAmetDNA SAH (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA SAM }, though this route is an order of magnitude slower.     

Improved Synthesis of Adenosine Cyclic 3′, 5′-Phosphoramidates via Anhydrides. Preparation of Adenosine Cylic 3′, 5′-(Rp) - and (p)-N-Methylphosphoramidate

Abstract

The two-step method for the preparation of adenosine cyclic 3′,5′-phosphoramidate diastereoisomers, which involves the activation of adenosine cyclic 3′,5′-monophosphate (1) with an acid chloride and in situ aminolysis of the anhydride intermediate (Bentrude, W.G.; Tomsaz, J. Synthesis 1984, 27; Bottka, S.; Tomasz, J. Tetrahedron Lett. 1985, 24, 2909), has been improved. The best yields were attained when 1 was reacted with 4.4 molar equivalents of phosphorus oxychloride in trimethyl phosphate at O°C for 3 h, and the solution of phosphorus oxychloride in trimethyl phosphate was pretreated with 0.5 molar equivalent of water at room temperature for 20 min. R _p and S _p diastereoisomers of adenosine cyclic 3′,5′-N-methyphosphoramidate and N,N-dimethylphosphoramidate have been synthesized under these experimental conditions.     

Localization and release mechanism of cellulases in Trichoderma reesei QM 9414

Summary A significant increase in the extracellular yield of -glucosidase was observed when Trichoderma reesei QM 9414 was cultivated on a cellulose medium containing chitin. Measurement of enzyme activities in the various fractions of the mycelium revealed that endoglucanase was truly extracellular while -glucosidase was cell wall bound. Treatment of Trichoderma mycelium with cell wall degrading enzymes (produced from Trichoderma) led to a release of -glucosidase from the mycelium. Apparently chitin, in the presence of cellulose, induces the synthesis of chitinase and other cell wall lytic enzymes which promote release of the intramural -glucosidase into the medium.     

Comparison between in vivo and in vitro enzyme activities in continuous and batch fermentations of Clostridium acetobutylicum

Summary In vitro activities of key enzymes and related parameters (ATP and ADP concentrations, intracellular pH (pH _i ), cell volume and the transmembrane pH) in various continuous and batch fermentations of Clostridium acetobutylicum were studied in order to investigate the regulation (genetic vs. enzyme level) of the solventogenesis process. In vitro activities varied significantly among an acidogenic (glucose limited) and three solventogenic (an iron limited, a CO gassed and a biomass recycle) continuous fermentations. However, in vitro enzyme activities did not correlate with in vivo specific production rates in continuous cultures indicating that solvent formation is regulated primarily at the enzyme level. Carbon monoxide (CO) gassing of an acidogenic continuous culture resulted in butyrate uptake without acetone formation due to inactivation of the acetoacetate decarboxylase by CO. In continuous, and to some extent in batch cultures, butyrate can be taken up via the reversal of the butyrate kinase and phosphotransbutyrylase pathway. Solvent formation in batch fermentations is both a result of enzyme induction and regulation. Acetone formation and the induction of acetoacetate decarboxylase occur simultaneously whereas both alcohol dehydrogenases are induced several hours before initiation of alcohol production. Finally, the levels of intracellular and related cell parameters (pH _i , pH, ATP and ADP concentrations) are discussed and related to the possible mechanisms of solventogenesis.