Synthesis and regulation of extracellular beta (1-3) glucanase and protease by cytophaga sp. In batch and continuous culture

Lytic enzyme systems with the ability to break whole cells of yeast are a mixture of several enzymes and virtually all contain beta(1-3)glucanases and some protease. It appears that the presence of these two enzyme activities is necessary to break the two layers of the rigid cell wall. The enzyme system of Cytophaga NCIB 9497 has a high activity towards the walls of yeast and also of bacteria. This article describes the production of this extracellular lytic enzyme system in batch and continuous culture-it was found to be inducible. The synthesis and regulation of the two main constituent enzymes, beta(1-3)glucanase and protease, have been investigated. The synthesis of beta(1-3)glucanase is regulated by bothinduction (by an unknown inducer) and catabolite repression. Highbeta(1-3)glucanase activities were obtained in continuous culture at low dilution rates over a narrow range (0.05-0.10 h(-1)), and there is evidence of the presence of more than one glucanase enzyme. Proteolytic activity appears subject to catabolite repression and made up of the activities of more than one protease enzyme. Productivity and enzyme concentration were increased several fold in continuous culture when compared to batch culture.     

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.     

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.     

Exploration of fungal spores as a possible storehouse of proteolytic biocatalysts

Studies were carried out to define the relation between enzyme production and fungal sporulation, in solid-state cultivation conditions of the filamentous fungus Aspergillus oryzae NRRL 2217 to get information on possible links between metabolite synthesis and differentiation phenomena. The efforts taken to explore the possibility for the presence of a neutral protease inside the spores of this fungus was to increase the overall enzyme yield. Results showed that the production of enzyme (neutral protease) and biomass (total protein) were synchronised, both reaching their respective maximum levels at 48 h of fermentation, and decreasing thereafter. Neutral protease synthesis was not related to sporulation. The spores produced were subjected to various permeabilisation procedures, and the increase in the levels of neutral protease was monitored. Mechanical shear was the sole technique that was able to disrupt spores but even this failed to increase enzyme titres, confirming the absence of intra-spore proteases.     

Lysosomal nature of hormonally induced enzymes in wheat aleurone cells

The subcellular distribution of the enzymes alpha-amylase, protease and ribonuclease in wheat aleurone layers after treatment with gibberellic acid was determined by differential centrifugation. Of the alpha-amylase 56% was precipitable from cell homogenates, indicating that it is a particulate enzyme. Similar results were recorded with protease. Particulate alpha-amylase showed distinct structural latency, and membrane-rupturing mechanical or chemical treatments were required to release the enzyme in an active form; the results were completely analogous to results with lysosomal enzymes found in animal tissues. The identification of the hormonally induced enzymes as lysosomal suggests that the hormonal mechanism may be more closely associated with extracellular enzyme synthesis rather than with nucleic acid metabolism.     

Regulation of Exocellular Proteases in Neurospora crassa: Induction and Repression of Enzyme Synthesis

Neurospora crassa strain 74A grown on Vogel's medium containing bovine serum albumin (BSA) as principal carbon source secretes proteolytic enzymes which appear in the culture filtrate. Low concentrations of sucrose (0.1%) are necessary for growth from conidia, as conidia will not germinate on BSA alone. Once growth is initiated, however, protease production begins and at 5 to 6 hr growth and enzyme production are parallel. Higher concentrations of sucrose (0.5-2%) repress protease synthesis. Other metabolizable materials (sugars, amino acids, peptide mixtures) also repress protease synthesis. Some sugars will not sustain growth but allow germination and full induction of protease in the presence of protein. A material found in culture fluids of cells during induction of protease synthesis when added to repressed cultures causes a five-fold increase in the amount of protease production, although this is still approximately half that of normally induced cells. This material appears to be produced by induced cells in as little as 2 hr of culture, which is before detectable levels of protease can be found. It is heat-stable, of low molecular weight, and is not a simple product of protein digestion by the N. crassa proteases.     

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 alkaline protease from an alkaliphilic actinomycete

The repression of alkaline protease synthesis from alkaliphilic actinomycete was studied by using glucose, peptone, yeast extract, KH2PO4 and amino acids; tyrosine, tryptophan, lysine, and arginine. There was a critical limit of stimulation of enzyme production by these components. Crude components such as molasses, wheat flour, and wheat bran were found to be effective for growth and enzyme production. The high level of enzyme production using agro-industrial by-products is commercially significant due to cheap nature of these sources. The findings are quite attractive, as only few actinomycetes, particularly alkaliphilic ones, have so far been explored for their enzymatic potential and regulation of enzyme synthesis.     

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.     

Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation,production and characterization

 Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions, is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers. The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa molecular mass range. Received: 7 June 1995/Received revision: 14 September 1995/Accepted: 20 September 1995     

Chemical Synthesis of 19F-labeled HIV-1 Protease using Fmoc-Chemistry and ChemMatrix Resin

HIV-1 protease (PR) has been extensively studied due to its importance as a target in AIDS therapy. The enzyme can be obtained via expression of its cloned gene in an appropriate system, or via chemical synthesis. We required a reliable source of fluorine-labeled HIV-1 protease for NMR studies. As our attempts to incorporate a labeling step in overexpression experiments in E. coli failed, we turned to chemical synthesis. Herein is described the first chemical synthesis of an active, 99 amino acid residue HIV-1 encoded protease using Fmoc-chemistry on a total PEG-based resin (CM resin), and labeled with ¹⁹F at the Phe residue. Also reported here are NMR studies of the labeled synthetic protein with a synthetic dimerization inhibitor.     

The use of crude lipase in deprotection of C-terminal protecting groups

A crude lipase, Newlase F, was used to remove C-terminal protecting groups from dipeptide esters. Hydrolysis of dipeptide n-heptyl esters with Newlase F was conducted in aqueous media containing acetonitrile. The optimum pH and temperature of lipase in Newlase F were 7.0 and 30 °C, respectively. Low level acetonitrile promoted the hydrolysis of dipeptide n-heptyl esters, while high level acetonitrile inhibited the hydrolysis. However, the protease activity in Newlase F was significantly inhibited by acetonitrile. Lipase in Newlase F worked better in a medium containing water-miscible organic solvents than in water-immiscible ones. N-terminal protecting groups were not affected by the protease in the crude enzyme. It was found that the protease in Newlase F did not hydrolyze amide bond with hydrophilic amino acids on either side under these conditions (pH 7.0, room temperature). Newlase F may consequently be used widely in the synthesis of peptide conjugates. The crude enzyme was immobilized on SBA-15 mesoporous molecular sieve. The lipase activity of immobilized preparation was more active on hydrolysis of C-terminal protecting groups and stable than the free enzyme. The immobilization also reduced the protease activity.     

Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii

AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.     

The action of cyclic-AMP on GA3 controlled responses I. Induction of barley endosperm protease and acid phosphatase activity by cyclic-3',5'-adenosine monophosphate

The effect of cyclic-AMP on the induction of protease and acidphosphatase activity in embryoless barley half-seeds was examined.Promotion of protease synthesis was observed with 5 mu cyclic-AMPand ADP. The protease induced by these compounds was similarto protease obtained with GA₃, in that the enzyme was dependenton ß-mercaptoethanol for enhanced activity and increasedprotease activity observed in the media after a 48 hr incubationwith cyclic-AMP and ADP was due to an increase in both the synthesisand release of this enzyme. Of a variety of adenine compoundsand nucleoside diphosphates tested only cyclic-AMP and ADP inducedprotease activity. When these same compounds were tested fortheir ability to induce acid phosphatase activity only cyclic-AMPwas consistently found to be active. The total amount of enzymeproduced as well as the rate of enzyme release was similar forhalf-seeds incubated with 5 mu cyclic-AMP or a saturating GA3concentration. Inhibition of the hormone or nucleotide inducedpromotion of acid phosphatase was observed when abscisic acidwas added to the incubation medium with one of these compounds.Some of the implications of these results are discussed. (Received May 11, 1971; )     

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.     

Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01

A mesophilic bacterium, Aeromonas veronii PG01, isolated from industrial wastes produced an extracellular thermostable organic solvent tolerant protease. The optimum condition for cell growth and protease production was pH 7.0 and 30 °C. The protease produced was purified 53-fold to homogeneity with overall yield of 32%, through ammonium sulphate precipitation, ion-exchange and gel permeation chromatography (GPC). The molecular weight, as determined by GPC–HPLC, was found to be about 67 kDa. SDS-PAGE revealed that the enzyme consisted of two subunits, with molecular weight of 33 kDa. The protease was active in broad range of pH from 6.0 to 10.0 with optimum activity at pH 7.5. The optimum temperature for this protease was 60 °C. The enzyme remained active after incubation at 50–60 °C for 1 h. This enzyme was stable and active after incubation with benzene and it was activated 1.3- and 1.5-fold by n-hexane and n-dodecane, respectively. This protease was inhibited completely by the classic metalloprotease inhibitor 1,10-phenanthroline and partially by the metal chelator EDTA but not by the serine protease inhibitor PMSF. The PG01 protease was found to contain 1.901 mol of zinc per mole of enzyme upon analysis by Inductively coupled plasma-optical emission spectroscopy. The thermostable and solvent tolerance property make it an attractive and promising biocatalyst for enzyme mediated synthesis.     

Regulation of a Sulfur-Controlled Protease in Neurospora crassa

Wild-type Neurospora crassa produces and secretes extracellular protease(s) when grown on a medium containing a protein as its principle sulfur source. Readily available sulfur sources, such as sulfate or methionine, repress the synthesis of the proteolytic activity. Preliminary characterization of the proteolytic enzyme shows it to have a molecular weight of about 31,000, a pH optimum of 6 to 9 with casein as substrate, and esterolytic activity against acetyl-tyrosine ethyl ester with a pH optimum of 8.5. The enzyme activity is completely inhibited by diisopropylfluorophosphate, partially inhibited by ethylenediaminetetraacetate, but unaffected by iodoacetate. The proteolytic activity is temperature labile and is reduced by 75% within 15 min at 60 C. Synthesis of the protease activity is induced by proteins, and to a lesser extent by large-molecular-weight polyamino acids, but not at all by small peptides or amino acid mixtures. During conidial out-growth, the protease(s) first appears at about 8 h and continues to increase while the cells are in an active growth phase. When a low concentration of sulfate is present, the protease(s) is not produced until about 18 h, suggesting that the sulfate must first be used by the cells before the protease is either synthesized or released.     

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.     

Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation.

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.