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.     

A gelatin-specific protease from hamster lung-derived cell cultures

Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited by p-chloromecuribenzoate, N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase.     

Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

A gelatin-specific protease from hamster lung-derived cell cultures

Summary Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited byp-chloromecuribenzoate,N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase. This research was supported by Program Project Grant HL-19717 from the National Heart, Lung, and Blood Institute, Grant AG 000-38-02 from the National Institute of Aging, and National Institute of Health Grant 5T32HL07035.     

Characterization of Salicola sp. IC10, a lipase- and protease-producing extreme halophile

In order to explore the diversity of extreme halophiles able to produce different hydrolytic enzymes (amylase, protease, lipase and DNAse) in hypersaline habitats of South Spain, a screening program was performed. A total of 43 extreme halophiles showing hydrolytic activities have been isolated and characterized. The isolated strains were able to grow optimally in media with 15–20% (w/v) total salts and in most cases, growth was detected up to 30% (w/v) total salts. Most hydrolase producers were assigned to the family Halobacteriaceae , belonging to the genera Halorubrum (22 strains), Haloarcula (nine strains) and Halobacterium (nine strains), and three isolates were characterized as extremely halophilic bacteria (genera Salicola, Salinibacter and Pseudomonas ). An extremely halophilic isolate, strain IC10, showing lipase and protease activities and identified as a Salicola strain of potential biotechnological interest, was further studied. The optimum growth conditions for this strain were 15–20% (w/v) NaCl, pH 8.0, and 37 °C. Zymographic analysis of strain IC10 detected the lipolytic activity in the intracellular fraction, showing the highest activity against p -nitrophenyl-butyrate as a substrate in a colorimetric assay, whereas the proteolytic activity was detected in the extracellular fraction. This protease degraded casein, gelatin, bovine serum albumin and egg albumin.     

Supernatant proteolytic activities of High-Five insect cells grown in serum-free culture

The proteolytic activity of High-Five insect cell culture supernatants was analysed using substrate gel electrophoresis (zymography). During growth in serum-free media, High-Five cells constitutively expressed and secreted proteases that were active on casein gel but not on gelatin or bovine serum albumin gels. Two main protease bands were visible at about 41–42 kDa and 32–33 kDa. By addition of various protease inhibitors in the incubation buffer, the proteases were identified as metalloproteases as complete and specific inhibition of the proteolytic activities was only obtained by 1,10-phenanthroline.     

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.     

Stability enhancement of hGM-CSF in transgenic<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> suspension cell cultures

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium, with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. Compared to the controls (4.72 μg/L), the extracellular hGM-CSF level could be increased to 39.78 μg/L with the addition of 5 g/L of gelatin.     

Characterisation and identification of spoilage psychotrophic Gram-negative bacteria originating from Tunisian fresh fish

The contamination of fresh fish by spoilage bacteria is undesirable, particularly when Gram-negative bacteria, which produce thermo-resistant protease and lipase, can grow. The purpose of the present work was, therefore, to isolate and identify psychrotrophic Gram-negative (Psy G(-)) bacteria, isolated from 80 samples of 12 species of wild and aquacultured fresh seafood, by biochemical and molecular methods using 16S rRNA gene sequencing. Twenty-eight identified strains were studied to evaluate their catalase, nitrate reductase, lipolytic and proteolytic activities, as well as growth ability, at different temperatures, pH and NaCl concentrations. Among 150 Psy G(-) strains, the most dominant species found were: Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damselae. All strains of Psy G(-) had catalase activity and were able to reduce nitrates to nitrites. Proteolytic activity on milk and on gelatin agar was demonstrated for the majority of the isolates. However, extracellular proteolytic activity as assessed by the azocasein method wasn’t very high in all the strains. Lipolytic activity, as assessed by the agar method, showed that 92.9 % of strains could hydrolyse egg yolk, against 82.1 % and 57 % that could hydrolyse Tween 20 and Tween 80, respectively.     

ACROSOMIC REACTION AND PROTEOLYTIC ACTIVITY IN THE SPERMATOZOA OF AN ANURAN AMPHIBIAN, LEPTODACTYLUS CHAQUENSIS (CEI)

Acrosomal reaction and acrosomal protease release in Leptodactylus chaquensis are correlated. When spermatozoa are included in gelatin films, the characteristic digestion halos around the sperm head can be observed only after the acrosomic reaction is accomplished. The addition of 10 mM EDTA to the gelatin films arrests the acrosomic reaction and no digestion halo is formed. Crude enzymatic preparations containing acrosomal proteases are equally active on gelatin and BAPNA substrates, irrespective of the presence of EDTA. Trypsin inhibitors (ovomucoid, lima bean and soybean) produce both diminution of the digestion halo and lowering of the activity of crude enzymatic preparations on gelatin or BAPNA substrates.     

北黄海沉积物可培养产蛋白酶细菌分离鉴定

【目的】揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源。【方法】分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性。【结果】从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas(69.9%)、Sulfitobacter(12.1%)和Salegentibacter(10.6%)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性。【结论】北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶。     

Enhanced expression of serine proteases during floral senescence in Gladiolus

Programmed cell death during senescence in plants is associated with proteolysis that helps in remobilization of nitrogen to other growing tissues. In this paper, we provide one of the few reports for the expression of specific serine proteases during senescence associated proteolysis in Gladiolus grandiflorus flowers. Senescence in tepals, stamens and carpels results in an increase in total protease activity and a decrease in total protein content. Of the total protease activity, serine proteases account for about 67-70% while cysteine proteases account for only 23-25%. In-gel assays using gelatin as a substrate and specific protease inhibitors reveal the enhanced activity of two trypsin-type serine proteases of sizes 75 kDa and 125 kDa during the course of senescence. The activity of the 125 kDa protease increases not only during tepal senescence but also during stamen and carpel senescence indicating that it is responsive to general senescence signals.     

Gene 21 protein-dependent proteolysis in vitro of purified gene 22 product of bacteriophage T4.

It has previously been shown that the product of gene 22 (P22) disappears completely from lysates of T4-infected bacteria during head formation and is not found in the finished phage. We show here that P22, as part of a phage head precursor, is subject to proteolysis in vivo. The only identifiable surviving fragments of this proteolysis may be the internal peptides, which are found inside the finished phage head.We further show that in vitro, head-defective lysates contain a protease activity highly specific for P22. The activity is dependent on the presence of wild-type gene 21 protein (P21). The protease is itself inactivated during the protein cleavages that accomplish capsid formation. The proteolytic activity is found associated with the defective heads produced by temperature-sensitive mutants in gene 23, but not in finished normal capsids.We have characterized this P21-dependent protease activity as it is exhibited in vitro.     

Comparative Characteristics of Human and Porcine Staphylococci and Their Differentiation in Burn Xenografting Procedures

Staphylococcus epidermidis from porcine skin differed from human cutaneous S. epidermidis in that the former strains were principally of the Baird-Parker biotype III group. The porcine-type strains were more proteolytic on casein and gelatin than were human strains, which were primarily of biotype II. Porcine strains were also elastolytic. Using supernatant fluids of broth cultures, the biotype II strains, but not the type III strains, were lipolytic in action on triolein. Both types of staphylococci were similar in enzymatic activities on Tween 80, egg yolk, and tributyrin. Elastase activity was not found in broth supernatant fluid of these bacteria. The porcine strains were retarded or inhibited from growing in media at pH 5.5. Action on casein agar followed by demonstration of elastase activity were used as markers to detect the porcine S. epidermidis strains in xenografts and on human burn wound grafting sites.     

Protease expression in the supernatant of Chinese Hamster Ovary cells grown in serum-free culture

The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B.     

Wall-associated processing of extracellular enzymes of Staphylococcus simulans biovar staphylolyticus

Staphylococcus simulans biovar staphylolyticus produces a staphylolytic glycylglycine endopeptidase (lysostaphin) and a micrococcolytic endo-β-N-acetylglucosaminidase (hexosaminidase) as proenzymes that are proteolytically processed through multiple intermediates to their mature forms by an extracellular sulfhydryl protease. Analysis of protease production by immunoblots using antiserum prepared against purified protease and by renaturing activity gels using gelatin as the substrate has revealed that the lysostaphin-processing protease also is produced as a proenzyme, which appears to be autocatalytically processed. Very little proprotease could be detected in supernatants from cultures of S. simulans biovar staphylolyticus, which suggested that the protein was being processed before it was released to the culture medium. Analysis of wall-associated proteins revealed that processing of proprotease occurred primarily in the cell wall. Furthermore, processing of prolysostaphin and prohexosaminidase also occurred in the cell wall matrix.     

Enzymes in the Dissolution Testing of Gelatin Capsules

Gelatin capsules are a widely used dosage form both for pharmaceutical drug products as well as dietary supplements. Gelatin in the presence of certain compounds, mainly aldehydes, or in high humidity and high temperature conditions can cross-link. Cross-linking involves covalent bonding of the amine group of a lysine side chain of one gelatin molecule to a similar amine group on another molecule. The covalent bonding is, for practical purposes, irreversible. Cross-linking results in the formation of a pellicle on the internal or external surface of the gelatin capsule shell that prevents the capsule fill from being released. In vitro dissolution testing of cross-linked gelatin capsules can result in slower release of the drug or no release at all. The data obtained by the Gelatin Capsule Working Group, created in the early 90s to investigate noncompliance of gelatin capsules, was used to establish the type and amounts of enzymes that can be added to the dissolution medium in the case of test failure to the presence of cross-linking in the gelatin. The two-tier dissolution testing was included in the US Pharmacopeia and it recommends the addition of pepsin (pH below 6.8) or pancreatin (pH above 6.8) to the medium depending on its pH. Pepsin shows good protease activity up to pH 4 and pancreatin above pH 6 leaving a gap where neither one has good activity. Possible proteolytic enzymes that could be used for the pH range 4–6.8 could be papain or bromelain.KEY WORDS: cross linking, gelatin capsules, two-tier dissolution testing     

Effects of calcium and magnesium on a 41‐kDa serine‐dependent protease possessing collagen‐cleavage activity

We report here the continued characterization of a 41‐kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin‐cleavage activity and an echinoderm‐specific collagen‐cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10‐phenanthroline (a metalloprotease inhibitor), and E‐64 (a thiol protease inhibitor) were without effect on the gelatin‐cleavage activity of the 41‐kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin‐cleavage activity. This result was confirmed in a quantitative gelatin‐cleavage assay using the water soluble, serine protease inhibitor [4‐(2‐aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin‐cleavage activity. Calcium and magnesium competed for binding to the 41‐kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41‐kDa species. J. Cell. Biochem. 80:139–145, 2000. © 2000 Wiley‐Liss, Inc.     

Extracellular Accumulation of Pyrroles in Bacterial Cultures

Aerobacter aerogenes, Paracolobactrum aerogenoides, Spirillum serpens, and gelatinous strains of Chromobacterium violaceum produced an extracellular, ether-soluble, Ehrlich-positive substance when grown in media prepared with gelatin hydrolysate. The substance has been tentatively identified as pyrrole-2-carboxylic acid. Both hydroxy-l-proline and allo-d-hydroxyproline have been shown to be precursors of the material. Gelatinous strains of Chromobacterium violaceum, but not the other positive cultures, produced two ether-insoluble pyrroles as well, the precursors of which occur in gelatin hydrolysate but have not yet been identified. The property of pyrrole formation in bacteria and its possible use as an aid in identification of bacteria was discussed.     

Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (~74, ~55, ~54, and ~37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.