Synthesis of the extracellular protease byBacillus pumilus

Bacillus pumilus synthesizes an oxtracellular protease during the stationary phase of growth when the intracellular protease level is rather low. The formation of the enzyme is blocked by chloramphenicol. A shift from batch to continuous cultivation is accompanied by a decreased enzyme level in the medium. The original concentration of the enzyme in the medium can be attained only after reverting to batch cultivation. The protease is not synthesized during growth in a mineral medium with glucose. Glutamic acid, arginine and ornithine are among the amino acids which stimulate the enzyme formation. Yeast extract enhances the formation of the protease, the active component of the extract being only the brown pigment with attached Fe³⁺ ions. The B-vitamins and other essential growth factors contained in the yeast extract are without effect on the protease formation. Free ferric ions also induce protease formation, the level being roughly proportional to the concentration of Fe³⁺ in the medium. The synthesis is also enhanced by Mn²⁺. Growth of the culture under oxygen limitation results in the suppression of protease formation.     

Regulation of the formation of protease inBacillus megaterium

During the growth of the asporogenous variant ofBacillus megaterium KM in medium containing NO₃ ⁻ as nitrogen source, the relative rate of extracellular protease synthesis is higher than in the presence of NH₄ ⁺. It approaches the relative rate of enzyme synthesis at the incubation of cells in nitrogen-free medium with glucose. This supports the suggestion that even amino acids which are synthesized endogenously slow down the protease production. In the postlogarithmic or stationary phase the protease production stops. The interruption of enzyme production does not appear as a result of insufficient aeration in a dense suspension, or of accumulation of amino acids or their metabolites in cells. The non-growing cells retain their ability to renew the enzyme synthesis when transferred into a fresh medium, even into a medium without nitrogen source. In the same way it is possible to “induce” the protease production, if Ca²⁺ is added to cells in the stationary phase when the population was grown in the Ca²⁺ free medium. The amount of enzyme produced at the expense of protein turnover by the non-growing populations is sufficient for the fast hydrolysis of exogenous protein in the medium and for assuring the influx of a sufficient amount of peptides into the cells. In such a case the growth of the culture is therefore very quickly renewed.     

Regulation of the formation of protease inBacillus megaterium

Protease is synthesized by the cultures growing in a glucose-containing mineral medium. However, it is formed even during incubation of the washed cells in a nitrogen free medium. The enzyme synthesis is decreased substantially by the addition of the individual amino acids or their mixture. Threonine, isoleucine, leucine and valine are the most inhibitory. Arginine, cysteine, glycine, lysine and tryptophan in concentrations of 10³ m do not inhibit the production of protease. The growth of the culture is also somewhat inhibited by threonine and isoleucine, the repression of protease being, however, much higher. Concentrations of 10³ m inhibit its synthesis by 80–90%. However, the enzyme activity is not influenced. The inhibition is caused byl,-isomers. Repression of the enzyme synthesis after the addition of threonine into the medium is much greater in a growing culture than in a culture starving in a nitrogen-free medium. However the level of free threonine in the pool is roughly the same in both growing and non-growing cultures. A mixture of 13 amino acids, which themselves are little inhibitory, suppresses the synthesis of protease much more than threonine or isoleucine. The inhibitory effect of the individual amino acids on the enzyme formation is apparently additive.     

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.     

Regulation of the formation of protease byBacillus megaterium

The formation of protease takes place in washed cells ofBacillus megaterium incubated in a nitrogen-free medium. The rate of enzyme synthesis is decreased much less than that of cell proteins as compared with growing cells. The synthesis of protease in a nitrogen-free medium requires the presence of glucose. The omission of glucose results in stopping of the enzyme formation and substantial decrease of the rate of protein synthesis. Protease is not synthesized when the washed cells are incubated in a phosphate, free medium. The incubation of the cells in a nitrogen-free medium results in a decrease of the concentration of amino acids in the pool. In a phosphate-free medium the content of free amino acids increases temporarily and decreases again later. When the culture grown in the medium containing threonine or threonine and isoleucine in addition to NH₄ ions is transferred into the medium without amino acids, no protease formation is found during derepression of enzymes synthesizing both amino acids. The cells grown in a medium containing casamino acids begin to form the enzyme after a short lag period when transferred into the medium containing NH₄ as a sole nitrogen source or into a nitrogen-free medium.     

Growth and development kinetics of Bacillus thuringiensis in batch culture

The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.     

Excretion of a protease by Serratia marcescens

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfurcontaining amino acids. The enzyme consists of one polypeptide chain. A molecular weight of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N--benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of diand tripeptides.Abbreviation SDS sodium dodecylsulfate     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.     

The production of protease by dense suspensions ofBacillus megaterium KM Sp−

The synthesis and secretion of extracellular protease was demonstrated during the incubation of dense susponsions of the asporogenicBacillus megaterium KM. The overall production of the enzyme by cells incubated with glucose in a nitrogen-free medium was found to be only slightly lower than that in the presence of an inorganic nitrogen source. The capacity to form protease decreased exponentially with increasing density of the bacterial suspension. The synthesis of the enzyme was interrupted after the exhaustion of glucose. A repeated exchange of the medium made it possible to reach relatively high and continuous production of protease for several hours. The total amount of extracellular proteins synthesized during incubation of the dense suspension in media with or without a nitrogen source was less than 2% of a total of newly formed proteins. The amount of these extracellular proteins was slightly lower in the absence of Ca²⁺ being considerably decreased when the dense suspension was incubated with chloramphenicol.     

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.     

Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated protein

Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully ¹³C‐labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Repression of alkaline protease in salt-tolerant alkaliphilic <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> strain Mit-1 under the influence of amino acids in minimal medium

A salt-tolerant alkaliphilic actinomycete (strain Mit-1) was isolated from Mithapur (Western Coast, Gujarat, India) and identified as Streptomyces clavuligerus. Based on 16S rRNA gene sequence (EU146061) homology, it was found to be related to Streptomyces sp. (AY641538.1). The strain secreted alkaline protease optimally at 5% NaCl and pH 9 during the early stationary phase and could utilize the amino acids methionine, alanine, leucine, phenylalanine, tyrosine, tryptophan, arginine, asparagine, histidine, and glutamic acid as the sole source of nitrogen. Above their threshold levels, these amino acids caused repression of alkaline protease production. Protease production with methionine (120 U/mL), histidine (140 U/mL), and aspartic acid (118 U/mL) was comparable to that with complex medium (130 U/mL). However, the production increased with an increasing number of different amino acids in the growth medium. Repression of protease production as influenced by the amino acids generated valuable information on enzyme synthesis in actinomycetes, as such data is scarce. Optimization of the conditions for enzyme production by actinomycetes in general, and in haloalkaliphilic actinomycetes in particular, appears to be an attractive proposition for biocatalysis.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

Regulation of protease production in Clostridium sporogenes

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Regulation of protease production in Clostridium sporogenes.

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Biochemical and physiological properties of an extracellular protease produced by Bacillus megaterium

A protease, excreted by a sporogeneous strain of B. megaterium, growing exponentially in a minimum glucose ammonium medium, was isolated. It is a neutral endopeptidase, stabilized by Ca⁺⁺, inhibited by o-phenanthroline, but not by di-isopropylfluorophosphate. The specificity, studied on insulin B-chain, glucagon, cytochrome c, and dipeptides substrates, indicated the need for a dipeptide backbone with both substituted amino and carboxyl groups. A requirement was observed for a nonpolar lateral chain in the amino acid whose amino group was involved in the peptide bond (Leu, Phe, Ala, He, Val). Rates of hydrolysis varied also with the amino acid whose carboxyl group was involved (e.g., His > Ser > Ala > Gly). In complex medium, supplemented with Yeast Extract, the biosynthesis of the protease was repressed during growth, but the same enzyme was excreted during sporulation. The repression was apparently of the same nature as that controlling sporulation during and after growth (e.g., repression by a mixture of amino acids or high concentration of glucose). An asporogeneous mutant showed a normal product ion of protease under all conditions, and a low intracellular protease turnover after growth. A mutant unable to produce protease showed a normal sporulation and a high protein turnover. This protease, here termed megapeptidase, seems to be a typical growth enzyme, not related to either the sporulation process or to the protein turnover after growth.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

Effect of amino acids and proteins on bacitracin and exoprotease synthesis by Bacillus licheniformis

Amino acids and proteins were found to produce different effect on the synthesis of bacitracin and exoprotease by Bacillus licheniformis 28 KA depending on the age of the cells. The enzyme synthesis was induced by amino acids and proteins only in the cells at the exponential growth phase. No correlation could be established between the antibiotic and proteolytic activities. The optimal protease synthesis was found in a medium with isoleucine whereas the antibiotic synthesis was optimal in a medium containing no amino acids.     

Production of Proteases from Brevibacterium Linens

Protease formation in submerged cultivations of Brevibacterium linens was studied. The effect of several proteinaceous materials on the production of proteolytic enzymes was investigated in mineral media containing 0.2% malt extract for bacterial growth. The addition (0.5%) of yeast extract or enzymatically hydrolyzed casein considerably increased the amount of protease formed, whereas ammonium salts supplied additionally in most cases had a repressive effect on enzyme formation. Furthermore, the kinetic of protease formation was determined in a highly instrumented fermenter system. Respiration activity indicated several phases of bacterial growth. Most of the proteolytic activity was synthesized during active growth; there was only a small increase in the stationary phase. A total proteolytic activity of 36 U/ml was formed in 24 hr. Concentration of α-amino nitrogen decreased steadily and ammonium ions accumulated during bacterial growth. Electrophoretic analysis revealed the occurrence of one leucine aminopeptidase (26 kDa monomer) and several proteases. There is a broad spectrum of proteolytic active proteins in the range of 11-66 kD which may be caused by some auto-degrading effects.     

Effect of amino acids on exoprotease synthesis by Bacillus thuringiensis

The influence of certain L-amino acids and their mixtures on the synthesis of exoprotease from Bacillus thuringiensis was studied. Physiological experiments showed that the mixture of 20 amino acids added to the artificial medium repressed the synthesis of exoprotease. Among the compounds studied there are both the compounds which stimulate the synthesis of exoprotease (glutamic and aspartic acids, glycine), and the compounds which repress the synthesis of the enzyme (proline, tryptophane, tyrosine, asparagine, serine, cystein). None of the amino acids caused a change in the exoprotease activity. It has been assumed that the repression of the protease synthesis in the presence of the amino acids is accomplished by ammonium ions, which are formed when using the amino acids of Bac. thuringiensis. The glutamine synthetase activity of cells was determined during the growth of Bac. thuringiensis both on a medium containing triptone and after the addition of certain amino acids to the cell suspension. The correlation between the influence of different amino acids on the synthesis of exoprotease and the glutamine synthetase activity was demonstrated.