The production of toxic protease by the entomopathogenous fungus Metarhizium anisopliae in submerged culture

The production of a toxic complex of proteolytic enzymes by Metarhizium anisopliae was evaluated with 29 nitrogen sources in modified Czapek-Dox medium in submerged cultures. The proteolytic complex is more constitutive than that of Beauveria bassiana and its production is influenced by the quality of complex natural media. The highest activity was attained with Galleria mellonella proteins. The proteolytic complex manifests proteolytic activity of two pH optima, 5.5 and 8.0. The ratio of these two activities differs markedly with the nitrogen source used, but the major proteolytic activity occurs at pH 5.5.     

Midgut protease activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri

We assayed the relative activities of midgut proteolytic enzymes in individuals of the fourth (L(4)) and fifth (L(5)) instar of Apollo larvae, inhabiting Pieniny Mts (southern Poland). The comparisons between midgut tissue with glicocalyx (MT) and liquid midgut contents with peritrophic membrane (MC) were made. Optimal media pHs of the assayed proteolytic enzymes in P. apollo midgut samples were similar to those of other lepidopteran species. Endopeptidases, as well as carboxypeptidases, digested effectively in alkaline environment, while aminopeptidases were active in a broad pH range. Trypsin is probably the main endoprotease (correlation with caseinolytic activity in MC of L(5) larvae: r=0.606; p=0.004); however, its activity was low as compared with that in other leaf-eating Lepidoptera. This suggests a minor role of trypsin and chymotrypsin in protein digestion in Apollo larvae, probably due to limited availability of the leaf proteins. Instead, due to very high carboxypeptidase A activity in midgut tissue, the larvae obtain exogenous amino acids either directly or from oligopeptides and glycoproteins. High and significant positive correlations between the enzyme activity and glucosidase as well as galactosidase activities strongly support this opinion.     

Neutral protease activity of Rous sarcoma (RSV) transformed chick embryo fibroblasts.

The proteolytic activities of normal, Schmidt-Ruppin Rous sarcoma virus (SR-RSV) transformed, and infected (RAV) chick embryo fibroblasts (CEF) have been measured by a highly sensitive technique using 3H-acetylated haemoglobin as a substrate.When all 3 types of CEF cells were maintained in serumless media, no differences were detected in the amount of pH 3-4 protease activity released into the media over a 24-h period, and only negligible amounts of pH 7-6 proteolytic activity were found. When normal, transformed, and infected cells were maintained in serumless media and later incubated with 3H-acetylate haemoglobin, a significant proteolysis of the haemoglobin, a 6-fold increase compared to the normal CEF cells, was associated only with plates containing SR-RSV-CEF cells. A fluorescent assay for peptides confirmed that SR-RSV-CEF cells have increased cell-associated proteolytic activity. The net surface charge of the transformed CEF cells was unchanged by maintenance in serumless media but the net surface negativity of the normal and RAV-CEF cells was significantly increased by incubation in media minus serum for 24 h. This suggests that normal CEF cells, maintained in media plus serum, have a substance masking their surface charge which is absent from the surface of transformed cells, possibly because of proteolytic degradation.     

Regulation of extracellular protease formation by Serratia marcescens.

Heavy cell suspensions of Serratia marcescens, when grown in gelatin-containing media, produce extracellular proteases which increase in specific activity in a linear fashion for 3 to 4h. During partial purification, a single peak of proteolytic activity was demonstrated by Sephadex G-100 chromatography. However, electrophoresis using 5% polyacrylamide gels discloses three proteolytically active bands. Evidence in favor of gelatin acting as an inducer of the 'proteolytic system' was provided by two observations. First, proteolytic activity is only present in media containing gelatin. Secondly, the addition of 10(-4) M rifampicin to cells growing in gelatin-containing medium plus an additional carbon source inhibits protease activity totally, but has no effect on growth. When glycerol is added to a growing cell suspension in gelatin-containing medium, growth increases, but protease specific activity decreases. This 'glycerol effect' is not due to an accumulation of active or inactive enzyme in association with the cell, nor to a decrease in the total number of proteases synthesized. Rather, glycerol, as other utilizable carbohydrates, exerts a repression which can be eliminated by 5 mM dibutyryl cyclic AMP.     

Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6

The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.     

Purification and partial characterization of a Nocardia brasiliensis extracellular protease.

Nocardia brasiliensis possess proteolytic activities that can be readily detected in a variety of media. In a modified formulation of a growth medium originally used for Streptomyces aureofaciens, N. brasiliensis was found to secrete proteolytic enzymes, one of which was capable of hydrolyzing casein. This enzyme was purified to homogeneity from cell-free culture filtrates of N. brasiliensis. The purification procedure included ion-exchange chromatography on carboxymethyl-Sepharose, gel filtration on Sephadex G-100, and affinity chromatography, using a hemoglobin-Sepharose resin. The molecular weight of the N. brasiliensis protease was found to be 25,000 by gel filtration and 35,000 by sodium dodecyl sulfate-discontinuous gel electrophoresis. The enzyme is inhibited by o-phenanthroline and 8-hydroxyquinoline-5-sulfonic acid but is not affected by EDTA. Average values for its kinetic parameters were 0.288 mumol of hemoglobin solubilized per min per mg of enzyme for Vmax and 0.76 mM for Km, using hemoglobin as the substrate.     

Dependence of Clostridium botulinum gas and protease production on culture conditions.

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.     

The protease problem in Neurospora. Variable stability of enzymes in aromatic amino acid metabolism.

A proteolytic activity isolated from Neurospora crassa is shown to be responsible for the variable stability observed in vitro for enzymes involved in aromatic amino acid metabolism. For example, the activity of kynurenine formamidase was insensitive to the action of this protease preparation over a 24-h period of incubation at 25 °C, whereas chorismate synthase, anthranilate synthase, kynureninase, and the five activities of the arom multienzyme system were inactivated during this time. Anthranilate synthase and two of the arom system activities (dehydroquinate synthase and shikimate kinase) were inactivated by the protease preparation within 2 h. Phenylmethanesulfonylfluoride and a specific proteolytic inhibitor from N. crassa prevented inactivation of these enzymes. Spontaneous loss of activity at 25 °C of purified samples of anthranilate synthase, dehydroquinate synthase and shikimate kinase was also prevented by the inhibitors. A method for purifying the inhibitor from N. crassa is described, and its use as a reagent in the analysis of proteolytic action is demonstrated.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells.

A sensitive method for measuring cell surface and secreted protease activity utilizing 3H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloracetic acid soluble 3H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3--1 times more proteolytic activity than the normal cells. Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.     

Efficient lactic acid production from high salt containing dairy by-products by Lactobacillus salivarius ssp. salicinius with pre-treatment by proteolytic microorganisms

Lactic acid bacteria have an inefficient proteolytic system. Therefore, cultivation media which may have high protein content are usually supplemented with yeast extract or protein lysates (peptones). These additives might be conveniently replaced by in situ treatment of the cultivation medium with proteolytic enzymes or proteolytic microbes. Lactobacillus salivarius ssp. salicinius, a lactic acid bacterium species that can grow at high salt concentration, was used to ferment lactic acid in cheese whey (with 3 gl(-1) whey protein content) and lactose mother liquor (90 gl(-1) lactose, 9 gl(-1) proteins, 30 gl(-1) minerals). The contribution of protease enzymes or proteolytic microbes to acid production by lactobacilli was examined. Efficient conversion of lactose to lactic acid was obtained in the presence of additional proteolytic activity. Fastest acid production was obtained with the addition of protease enzymes. However, almost equally efficient acid production was obtained by treating the medium with Bacillus megaterium. The results show that fast and complete conversion of lactose to lactic acid can be obtained in dairy by-products without expensive additives.     

Biosynthesis of alpha-amylase and protease by Streptomyces olivaceus 142. I. Regulation of alpha-amylase activity.

Streptomyces olivaceus 142 produces amylase in the logarithmic phase of growth of the culture. The synthesis of the enzyme is induced by maltose and starch. In the case of maltose the synthesis is induced by a contaminating compound, probably being a higher than maltose polymer of glucose. The synthesis of amylase is negatively controlled by catabolic repression. The level of the activity of the enzyme depends not only on the biosynthesis but also on it proteolytic degradation.     

Biosynthesis of protease from Lactobacillus paracasei: kinetic analysis of fermentation parameters

Fifteen strains of Lactobacillus species, isolated from different samples of curd were screened for their ability to produce more extracellular protease. The proteolytic activities of these strains based on casein hydrolysis showed a variation of 1.26-5.80 U ml(-l), with Lactobacillus IH8 showing the maximum activity and was identified as L. paracasei. Different cultural conditions for enhanced production of protease by L. paracasei were optimized. The optimal conditions for production of the enzyme were an incubation temperature of 35 degrees C and a medium pH of 6.0. The maximum proteolytic activity of L. paracasei (7.28 Uml(-1)) was achieved after 48 h of cultivation. The kinetic parameters such as product yield (Yp/x,), growth yield (Yx/s), specific product yield (qp) and specific growth yield (qs) coefficients also revealed that the values of experimental results were kinetically significant.     

Staining for protease activity on polyacrylamide gels

A method for staining electrophoresis gels for proteolytic activity of both serine and sulfhydryl enzymes is described. The gels are incubated in the presence of azocoll powder and then scanned.     

Detection of extracellular protease in Mucor species

The fungi are characterized by their abilities to produce and secrete enzymes to the external environment. The species of genus Mucor are a group of fungal microbes with important biotechnological potential, which are responsible for production of industrial enzymes. This work evaluated the ability of protease production in twelve species of genus Mucor. The strains were kept for 120 h under the incubation temperature of 28 degrees C on a shaker at 120 rpm. The detection of proteolytic activity was evaluated in all species, the higher activity was detected in Mucor racemosus Fres. f. chibinensis (Neophytova) Schipper.     

Variants of Myxococcus virescens Exhibiting Dispersed Growth. Growth and Production of Extracellular Enzymes and Slime

The growth of two strains of Myxococcus virescens exhibiting dispersed growth was followed in casamino acids (N III-C) media and casitone media. The changes in optical density, pH, pigmentation as well as the secretion of bacteriolytic and proteolytic enzymes, DNA and polysaccharides during growth were recorded. In both media the bacteria grew exponentially with a generation time of 4 (casitone) and 20 hours (N III-C) respectively. The maximal cell mass was about 4 times higher in casitone than in casamino acids media. The amounts of bacteriolytic enzymes produced by the two strains in N III-C medium were different but in casitone medium they were about equal and considerably higher. The maximal values of proteolytic enzymes were about the same in both media and always occurred later than the bacteriolytic maxima. Both activity peaks appeared before the phase of decline. The polysaccharide production reached a maximum during the stationary growth phase in both media. A higher value was reached during growth in casitone medium than in N III-C medium. During the phase of decline a second increase of polysaccharide in the medium appeared. No DNA could be detected in the cell-free solutions until the beginning of the phase of decline.     

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.     

Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.     

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.     

Dependence of Clostridium botulinum gas and protease production on culture conditions

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.