Enniatin B synthesis by a Fusarium sambucinum Fuck culture

Three fungal strains belonging to the genus Fusarium Lk. ex. Fr. (F. sambucinum Fuck. 52377, F. avenaceum (Fr. Sacc.) 52311, F. gibbosum App. et. Wr. emend Bilai 52021) whcih form 800-1200 mg of enniatin B per litre during submerged cultivation have been selected. The morphology of F. sambucinum 52377 in the course of growth and production of enniatin B on the selected medium is described. The maximum accumulation of the product is found at the stationary growth phase. The active accumulation of fatty inclusions during this period suggests the participation of metabolism of fatty acids in the biosynthesis of enniatin B.     

Enniatin Production by Fusarium Strains and Its Effect on Potato Tuber Tissue

Several Fusarium strains produce the cyclohexadepsipeptide enniatin, a host-nonspecific phytotoxin. Enniatins are synthesized by the 347-kDa multifunctional enzyme enniatin synthetase. In the present study, 36 Fusarium strains derived from a wide range of host plants were characterized with respect to enniatin production in different media. Thirteen of these strains produced enniatins on one or more of these media. To determine whether enniatin production affected virulence, an assay on potato tuber tissue was performed. Seven enniatin-producing and 16 nonproducing strains induced necrosis of potato tuber tissue, so that enniatin synthesis is not essential for the infection of potato tuber tissue. The application of a mixture of enniatins to slices of potato tuber, however, caused necrosis of the tissue. Therefore, enniatin production by the enniatin-synthesizing strains may affect their pathogenicity. The enniatin synthetase gene (esyn1) of Fusarium scirpi ETH 1536 was used as a probe to determine if similar sequences were present in the strains examined. In Southern blot analyses, DNA sequences hybridizing with the esyn1 probe were present in all but two of the strains examined. In some cases, enniatin-nonproducing strains had the same hybridization pattern as enniatin producers.     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono-and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1–1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25–30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca²⁺, Mg²⁺, and Mn²⁺ stimulated biosynthesis of proteinase in the late stationary phase (by 20–60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Biosynthesis of chitin by particulate fractions from Cunnighamella elegans.

The enzyme chitin synthetase (UDP-acetylaminodeoxyglucosyl transferase, EC 2.4.1.16) in Cunninghamella elegans has been investigated. The enzyme was present in the microsomal, cell wall, mitochondrial and the soluble cytoplasmic fraction of the mycelium, with the former having the highest specific activity. The properties of the enzyme in this fraction were investigated; the Km for UDP GlcNAc was 1.23 mM and 2.08 mM GlcNAc in the presence of 1 mM UDP GlcNAc. The temperature optimum was between 26 degrees and 29 degrees C and maximal activity was at pH 6.25. Mg++ ions had no effect on chitin synthesis, but soluble chitodextrins inhibited the enzyme. The production of chitin synthetase was correlated with the growth of the fungus, maximum activity being found during the late exponential phase of growth. Chitin was confirmed as the sole product of enzyme action, by digestion with chitinase.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth.     

Selective synthesis of depsipeptides by the immobilized multienzyme enniatin synthetase

Summary The multifunctional enzyme enniatin synthetase was immobilized by adsorption to propyl agarose. The immobilized multienzyme retained 45% of the activity of the free enzyme; an operational half-life of about 15 h was estimated. Selective synthesis of several different enniatin homologues was achieved with propyl agarose-bound enniatin synthetase. In addition to enniatin A, B, and C formation, a selective synthesis of non-naturally occurring depsipeptides, containing norvaline, norleucine, or -aminobutyric acid as sole amino acid moieties, was observed.     

Influence of ferric iron on gene expression and rhamnolipid synthesis during batch cultivation of Pseudomonas aeruginosa PAO1

Bioprocesses based on sustainable resources and rhamnolipids in particular have become increasingly attractive in recent years. These surface-active glycolipids with various chemical and biological properties have diverse biotechnological applications and are naturally produced by Pseudomonas aeruginosa. Their production, however, is tightly governed by a complex growth-dependent regulatory network, one of the major obstacles in the way to upscale production. P. aeruginosa PAO1 was grown in shake flask cultures using varying concentrations of ferric iron. Gene expression was assessed using quantitative PCR. A strong increase in relative expression of the genes for rhamnolipid synthesis, rhlA and rhlC, as well as the genes of the pqs quorum sensing regulon was observed under iron-limiting conditions. Iron repletion on the other hand caused a down-regulation of those genes. Furthermore, gene expression of different iron regulation-related factors, i.e. pvdS, fur and bqsS, was increased in response to iron limitation. Ensuing from these results, a batch cultivation using production medium without any addition of iron was conducted. Both biomass formation and specific growth rates were not impaired compared to normal cultivation conditions. Expression of rhlA, rhlC and pvdS, as well as the gene for the 3-oxo-C12-HSL synthetase, lasI, increased until late stationary growth phase. After this time point, their expression steadily decreased. Expression of the C4-HSL synthetase gene, rhlI, on the other hand, was found to be highly increased during the entire process.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

Ammonium repression of cephalosporin production by Streptomyces clavuligerus

Production of beta-lactam antibiotics took place during growth of Streptomyces clavulgerus in chemically defined medium. The specific activities of isopenicillin N synthetase ("cyclase"), isopenicillin N epimerase, and deacetoxycephalosporin C synthetase ("expandase") increased during the exponential phase of growth. Specific cephalosporin productivity during fermentation followed a similar pattern, reaching a maximum near the end of the growth phase and decaying rapidly in the stationary phase. Ammonium chloride depressed cephalosporin production, presumably as a result of repression of cyclase and expandase formation, but not of epimerase. No inhibitory effects on enzyme activity by ammonium were found. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] prevented the repression of cyclase and markedly stimulated cephalosporin production. Cephamycin C and, in smaller amounts, O-carbamoyldeacetylcephalosporin C were the only cephalosporins detected. Growth with ammonium resulted in lower titers of both compounds, and did not change the relative proportion of each. The correlation found between cephalosporin productivity and cyclase specific activity in different media suggests that formation of this enzyme may be the rate-limiting step in the pathway.     

Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.     

Lipopeptide Production in Pseudomonas sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System

Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.     

The life style of Aureobasidium pullulans and the multiple forms of its polygalacturonase

The life style of Aureobasidium pullulans on pectin medium and its production of extracellular polygalacturonases are closely related. Polygalacturonases with random action pattern (EC 3.2.1.15) were formed in the first phases of cultivation, whereas exopolygalacturonases (EC 3.2.1.67) with terminal action pattern on pectin were produced during the whole growth of this yeast-like fungus. The production and inactivation of individual enzyme forms during cultivation were strongly dependent on the pH value of the pectin medium. Various kinds of stress can support the prolongation of the phase of endo-acting enzyme production, as well as the increase of their activity.     

Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.     

The Influence of Oxygen on the Production of Volatile Compounds by Dipodascus aggregates

The production of volatile compounds by Dipodascus aggregatus was studied in relation to the oxygen concentration in the medium. Oxygen concentration was determined with a Clark oxygen electrode and volatile compounds in the atmosphere above the culture by a gas chromatographic technique. Shake cultures of the fungus in its stationary phase of growth were very sensitive to a decrease in oxygen concentration in the presence of residual glucose. Anaerobic conditions induced production of volatile compounds that continued for many hours. The pattern of production of volatile compounds observed under conditions of low oxygen concentration during the stationary phase of growth differed from that obtained under aerobic conditions during the exponential phase of growth.     

Biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Kinetics of cellulase production intrichoderma viride on microcrystalline cellulose

During the cultivation of a wild strain ofT.viride on microcrystalline cellulose the synthesis of cell-bound FP cellulases precedes cell growth. During the growth they are released into the medium as extracellular enzymes. The rate of synthesis of extracellular FP cellulases increases during cell growth, reaching a maximum at the beginning of transition to the stationary phase when the cell growth rate decreases. In contrast to extracellular enzymes, the rate of synthesis of bound cellulases during active growth is almost constant. In the stationary phase the rate of synthesis of both FP cellulases drops sharply, ceasing well before cell lysis sets in and before the maximum level of extracellular cellulases is attained.     

The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.     

Cellular Localization of Acetyl-Coenzyme A Synthetase in Yeast

In cells of Saccharomyces cerevisiae grown with glucose in standing cultures, the microsomal fraction had the highest specific activity for acetyl-coenzyme A synthetase and contained the greatest fraction of the total activity regardless of when the cells were harvested during growth. The addition of acetate did not affect the distribution of the enzyme, nor did subsequent aeration of such cells in phosphate buffer even in the presence of glucose, acetate, or succinate. In cells grown aerobically, however, the microsomal fraction had the highest specific activity and the greatest fraction of the total activity only until the cells reached the stationary phase. After this time, most of the activity was associated with the mitochondrial fraction. Finally, 3 or 4 days after inoculation, this fraction appeared to lose most of the enzyme to the microsomal and soluble fractions. Chloramphenicol, at concentrations that interfered with respiration but not with fermentation, prevented the association of acetyl-coenzyme A synthetase with the mitochondrial fraction in aerated cells, but it did not appreciably affect the large increases in enzyme activity observed during aerobic incubation. Cells grown with glucose under strict anaerobic conditions contained barely detectable amounts of acetyl-coenzyme A synthetase.