Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.     

Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity

Several thermophilic anaerobic bacteria with keratinolytic activity growing at temperatures between 50 degrees C and 90 degrees C were isolated from samples collected on the island of S?o Miguel in the Azores (Portugal). On the basis of morphological, physiological, and 16S rDNA studies, the isolate 2KXI was identified as a new species of the genus Thermoanaerobacter, designated Thermoanaerobacter keratinophilus. This strain, which grows optimally at 70 degrees C, pH 7.0, and 0.5% NaCl, is the first member of the genus Thermoanaerobacter that has been described for its ability to degrade native keratin. Around 70% of native wool was solubilized after 10 days of incubation under anaerobic conditions. The strain was shown to possess intracellular and extracellular proteases optimally active at 60 degrees C, pH 7.0, and 85 degrees C, pH 8.0, respectively. Keratin hydrolysis was demonstrated in vitro using a sodium dodecyl sulfate gel containing feather meal. The extracellular protease responsible for breaking down keratin fibers was purified to homogeneity in only one step by applying hydroxyapatite column chromatography. The enzyme belongs to the serine-type proteases and has a molecular mass of 135 kDa.     

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Differential expression of proteases in human gestational tissues before, during and after spontaneous-onset labour at term.

A number of tightly regulated proteolytic enzyme systems, including the plasminogen activation cascade and matrix metalloproteases, play integral roles in the remodelling of extracellular matrices during pregnancy and parturition. This study assessed these labour-associated changes in protease activity in human gestational tissues. Amnion, choriodecidua and placenta collected from women before (at caesarean section, not in labour), during (at caesarean section, in labour) and after (spontaneous-onset labour, normal vaginal delivery) labour were examined on gelatin-substrate SDS-PAGE zymography. All tissues displayed major 55 kDa plasminogen-dependent activity that was abolished by the serine protease inhibitors (10 mmol phenylmethyl-sulphonylfluoride l-1, 100 mmol epsilon aminocaproic acid l-1, 1 mmol Glu-Gly-Arg chloromethylketone l-1). The enzymic activity was identified as urokinase plasminogen activator on the basis of its co-migration with reference standard and western blot analysis, and did not vary with labour status. An additional protease with an apparent molecular mass of approximately 90 kDa was detected in all tissues. Densitometric measurement of these tissues showed a significant (P < 0.05) increase in this enzyme activity with labour onset. Heavy metal chelators (1 mmol 1.10 phenanthroline l-1 and 10 mmol EDTA l-1) selectively blocked the 90 kDa activity, consistent with the proposal that it is a metalloprotease. Co-migration with reference standard and western blot analysis confirmed the identity of this protease as the matrix metalloprotease 9 (MMP-9). Immunoreactive MMP-9 protein was also significantly (P < 0.05) increased during and after labour compared with before labour in all tissues examined. It is proposed that the upregulated expression of MMP-9 is involved in fetal membrane rupture and placental separation during and after labour onset, respectively. In conclusion, the regulated repertoire of protease activities expressed by human gestational tissues implies an important role for matrix-degrading enzymes during human parturition.     

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Dactylella shizishanna

AIMS: To evaluate the production of an extracellular serine protease by Dactylella shizishanna and its potential as a pathogenesis factor. METHODS AND RESULTS: An extracellular alkaline serine protease (Ds1) was purified and characterized from the nematode-trapping fungus D. shizishanna using cation-exchange chromatography and hydrophobic interaction chromatography. The molecular mass of the protease was approximately 35 kDa estimated by SDS-PAGE. The optimum activity of Ds1 was at pH 10 and 55 degrees C (over 30 min). The purified protease could degrade purified cuticle of Penagrellus redivivus and a broad range of protein substrates. The purified protease was highly sensitive to phenylmethyl sulfonyl fluoride (PMSF) (0.1 mmol l(-1)), indicating it belonged to the serine protease family. The N-terminal amino acid residues of Ds1 are AEQTDSTWGL and showed a high homology with Aozl and PII, two serine proteases purified from the nematode-trapping fungus Arthrobotrys oligospora. CONCLUSIONS: Nematicidal activity of D. shizishanna was partly related to its ability to produce extracellular serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: In this report, we purified a new serine protease from D. shizishanna and provided a good foundation for future research on infection mechanism.     

Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae.

Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity.     

Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris

We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.     

A 25-kDa Serine Peptidase with Keratinolytic Activity Secreted by <Emphasis Type="Italic">Coccidioides immitis</Emphasis>

Coccidioides immitis is the causative agent of coccidioidomycosis, a systemic mycosis that attacks humans and a wide variety of animals. In the present study, we showed that the C. immitis mycelial form is able to release proteolytic enzyme into the extracellular environment. Under chemically defined growth conditions, mycelia secreted seven distinct polypeptides ranging from 15 to 65 kDa and an extracellular peptidase of 25 kDa. This enzyme had its activity fully inhibited by phenylmethylsulphonyl fluoride, a serine peptidase inhibitor. Conversely, metallo, cysteine, and aspartyl peptidase inhibitors did not alter the 25-kDa enzyme behavior. This extracellular serine peptidase was able to degrade keratin, a fibrous protein that composes human epidermis. Additionally, this peptidase cleaved different protein substrates, including gelatin, casein, hemoglobin, and albumin. Curiously, an 18-kDa serine peptidase activity was evidenced solely when casein was used as the co-polymerized protein substrate into the gel. The existence of different secreted peptidases could be advantageous for the adaptation of C. immitis to distinct environments during its complex life cycle.     

Degradation of chicken feathers by Chrysosporium georgiae

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin–salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8~pH at 30 °C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin–salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracelluar keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermophilic protease production by Streptomyces sp. 594 in submerged and solid-state fermentations using feather meal

AIMS: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme. METHODS AND RESULTS: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete. CONCLUSIONS: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.     

Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus

Bacillus sp. P45, isolated from the intestine of the Amazon basin fish Piaractus mesopotamicus, showed proteolytic activity when grown on skimmed milk and feather meal agar plates. The keratinolytic potential of this strain was evaluated on whole feather broth and human hair broth. Bacillus sp. P45 degraded almost 90% of chicken feathers after 72 h of submerged cultivation on whole feather broth, and the production of extracellular proteases was observed. The formation of thiol groups was also detected during growth, indicating the contribution of sulphitolysis to the efficient hydrolysis of feather keratin. Nevertheless, Bacillus sp. P45 was unable to degrade hair keratin, possibly due to the conformational diversity of this substrate in comparison to feather keratin. Additionally, preliminary results demonstrated that this strain might be utilized in the degradation of recalcitrant collagen-containing wastes. The keratinolytic character of Bacillus sp. P45 might be utilized in environmental-friendly processes such as bioconversion of waste feathers, representing an alternative way of waste management that could lead to the production of value-added products such as microbial biomass, protein hydrolysates and proteolytic enzymes.     

The 84-kDa Form of Human Matrix Metalloproteinase-9 Degrades Substance P and Gelatin

Abstract: Matrix metalloproteinase-9 (MMP-9) is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix. Because collagen is not readily localized where neurons have been shown to produce MMP-9 in the human brain, the ability of this enzyme to degrade bioactive peptides was investigated with representative tachykinin peptides [substance P (SP), neurokinin A, neurokinin B, and kassinin]. Latent MMP-9 (94 kDa) was purified from the human cell line HL-60 and converted to an intermediary active form (84 kDa) with p -aminophenylmercuric acetate. This active form of MMP-9 degraded SP with a k _cat/ K _m of 170 m M ⁻¹ min⁻¹, which is 30-fold greater than the previously reported value for a representative collagen-derived peptide. The major digestion products were identified as SP_1–6 and SP_7–11, which were derived from cleavage of the Gln⁶-Phe⁷ bond. Minor products were also generated from cleavage of the Gly⁹-Leu¹⁰ bond. The other representative tachykinin peptides were cleaved at rates >10-fold slower than that of SP. The 84-kDa peptidase was also active as a gelatinase. Longer treatment of MMP-9 with p -aminophenylmercuric acetate caused the conversion of the 84-kDa enzyme to the established 68-kDa active form; however, the rate of SP degradation did not increase. Because MMP-9 is produced by neurons of the CNS, these results suggest a possible regulatory role for the enzyme in intercellular communication by altering the availability of bioactive peptides.     

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.     

Matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-beta fibrils (fAbeta), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Abeta (sAbeta) could degrade fAbeta as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAbeta and that this ability is not shared by other sAbeta-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAbeta was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAbeta breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAbeta were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Abeta that are cleaved during its degradation. Only MMP-9 digests contained fragments (Abeta(1-20) and Abeta(1-30)) from fAbeta(1-42) substrate; the corresponding cleavage sites are thought to be important for beta-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAbeta and may contribute to ongoing clearance of plaques from amyloid-laden brains.     

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage

AIMS: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk. METHODS AND RESULTS: Thirty-seven isolates of Ps. fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively. The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol x l(-1) calcium chloride at 20 degrees C. The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol x l(-1) EDTA. They were heat stable and retained activity even after treatment at 121 degrees C for 20 min. One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration. Attempts to purify the lipases proved unsuccessful. CONCLUSIONS: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments. However, the smaller proteases have not been described previously. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples.     

Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties

Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca²⁺ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The K_m values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P₁ position.     

Identification of serine proteases from Leishmania braziliensis

Leishmania (V) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nalpha-benzoyl-L-arginine ethyl ester hydrochloride and Nalpha-p-tosyl-L-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-L-phenyl-alanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.     

Novel extracellular proteolytic activity in Pediococcus acidilactici ATCC 8042

Proteolytic systems are common in lactic acid bacteria, but there are few reports about proteases or peptidases in the genus Pediococcus. To evaluate the presence of these types of enzymes, Pediococcus acidilactici ATCC 8042 was cultured in MRS broth. Supernatants collected during the log phase showed proteolytic activity towards an elastin dispersion when assayed using a spectrophotometer. Zn2+ showed a stimulatory effect, and the proteolytic activity reached its maximum when 200 mmol/L NaCl was included in the reaction buffer. On the other hand, activity was reduced when 5 mmol/L EDTA, 10 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L 1,10-phenanthroline were used or when the sample was heat treated. Zymograms showed two different proteolytic bands when gelatin was used as a substrate (>200 and 107 kDa), but only the higher molecular mass band was detected when casein or elastin was used. The gelatinolytic activity was not detected with zymograms of the 107 kDa band, which was the one inactivated by heat treatment. The use of a renaturing SDS-PAGE gel with embedded Micrococcus lysodeikticus cells allowed for the detection of a band with peptidoglycan hydrolase activity migrating at about 110 kDa. This activity was lost when 10 mmol/L EDTA was added to the renaturing buffer. Therefore, Pediococcus showed at least three different extracellular enzymes that were produced during the logarithmic growth phase and acted on peptide substrates. Each showed different substrate specificity, ion requirements, and thermostability.