Detection and characterization of extracellular proteases in Butyrivibrio fibrisolvens H17c

Summary The detection and approximate molecular weights of extracellular serine protease isoenzymes produced by Butyrivibrio fibrisolvens H17c were determined by gelatin-PAGE. Nine bands of protease activity with apparent molecular weights of approximately 101000, 95000, 87000, 80000, 76000, 68000, 63000, 54000 and 42000 were detected after gelatin-PAGE of supernatants from exponential phase cultures. A tenth serine protease band with an apparent molecular weight of approximately 32000 was detected in stationary phase cells. The activities of all ten protease bands were inhibited by a serine protease inhibitor but their activities were not affected by inhibitors of trypsin-like enzymes or metallo-, sulphydryl-and carboxylproteases. The activity of all ten exoprotease bands was optimal between pH 6.0 and 7.5. The ten exoprotease bands were only detected in media containing trypticase or casamino acids as nitrogen sources. Production of the ten protease bands was not affected by the carbohydrate source.     

Metallo-protease activity increases prior to ovulation in brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) follicle walls

Proteolytic activity was measured in the follicle wall surrounding oocytes from brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) by use of two different protease assays: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (substrate-SDS-PAGE) and a chromogenic synthetic peptide for type I collagen. In brook trout follicle walls, substrate-SDS-PAGE studies demonstrated that the activity of two proteolytic enzymes (80 kDa and 66 kDa) increased significantly before ovulation. The 80 kDa enzyme decreased significantly after ovulation whereas the 66 kDa enzyme remained elevated following ovulation. In yellow perch follicle walls, substrate-SDS-PAGE studies demonstrated that the activity of the major protease (66 kDa) increased before ovulation and remained elevated after ovulation. A chromogenic synthetic peptide was used to assay collagenolytic activity in follicle walls of brook trout and yellow perch. This assay revealed that collagenolytic activity increased significantly in both species before ovulation and remained elevated after ovulation. These findings suggest that metallo-proteases are involved in digesting the follicle wall in teleosts before and after ovulation.     

Proteolytic cleavage of IgG and other protein substrates by Dirofilaria immitis microfilarial enzymes

Proteases were detected in aqueous extracts of Dirofilaria immitis microfilariae. Enzymes within the extract were capable of hydrolyzing Azocoll, a general protease substrate, at pH's 7, 8, and 9. Sensitivities to a variety of protease inhibitors indicated that multiple azocollytic enzymes were present in the extract, most prominent of which appear to belong to the serine class of proteases. By incorporating various substrates into the matrices of polyacrylamide gels, 2 SDS-resistant, mercaptoethanol-sensitive proteases in the MF extract were identified at 22 and 76 kDa. These proteases showed differential abilities to digest casein, fibrinogen, hemoglobin, and IgG. The MF extract hydrolyzed radiolabeled IgG into 8-10-kDa fragments following a 20-hr incubation. A similar degree of digestion was observed in 2 hr when viable microfilariae were used. The potential significance of these proteases in the evasion of host effector mechanisms is discussed.     

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.     

Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

Proteolytic enzymes in wheat leaves were studied using zymographic detection of enzyme activities on one-way (1D) SDS-polyacrylamide gels and two-dimensional (2D) ones, on which protein samples were isoelectrofocused prior to PAGE separation. Gelatin of concentration 0.1 %, copolymerized into SDS-PAGE gels, digested by active proteinases enabled detection of those enzymes. On 1D gels, seven bands were seen and assigned to particular families through the use of specific inhibitors. Metalloproteinases inhibited by 20 mM EDTA were detected as 150 kDa band; aspartic proteinases were assigned to 115–118 kDa double band by using 25 mM pepstatin; 10 mM phenylmethylsulfonyl fluoride used for detection of serine proteinases pointed to band of 70 kDa and finally due to 10 μM E-64 inhibitor, cysteine proteinases of 37 and 40 kDa were detected. On 2D gels, additional separation according to protein isoelectric points enabled detection of proteinase isoforms. In the range of 4.5–6 pI, six metalloproteinases as well as ten aspartic proteinases were visible, ten serine- isoforms of pI 4.5–6.8 and four cysteine proteinases of 4.5–5.0 pI were found. Presented results were detected as reproducible results observed at least in four independent biological replications.     

Occurrence of two endoproteinases in wheat leaf intercellular washing fluid

The presence of endoproteinases in the intercellular washing fluid of the first wheat ( Triticum aestivum ) leaf 13 days after sowing was investigated. Two activities were detected after separation of the intercellular fluid proteins by SDS-PAGE using slab gels containing gelatin as substrate. Their sizes were 70 and 100 kDa. Both enzymes hydrolyzed gelatin and casein. They also seem to be involved in the degradation of a 40 kDa protein component of the intercellular fluid. These results indicate that both enzymes are endoproteinases. They were also obtained from leaves previously sumitted to intercellular fluid extraction. However, the 100 kDa enzyme yield was low, indicating that it is mainly located in the intercellular washing fluid. Assays performed with either specific substrates or inhibitors indicate that both enzymes are serine proteinases.     

Characterization of a serine protease activity in Sarcocystis neurona merozoites

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.     

Setaria cervi: in vitro released collagenases and their inhibition by Wuchereria bancrofti infected sera

In vitro released products of adult Setaria cervi females, microfilariae and extracts showed considerable amounts of collagenase activity. On the basis of per mg protein released in vitro, the products of both microfilariae and adult females exhibited comparable activity but this was much higher than that of extract of microfilariae and adult females. Two collagenase enzymes with molecular masses of 50 kDa and 70 kDa were separated using DEAE-sepharose CL6B and Sephadex G-100 column chromatography. The 50 kDa and 70 kDa collagenase exhibited pH optima of 5.2 and 7.0, respectively. Considering specific activity, the 50 kDa enzyme was found to contribute about ten times more collagenase activity as compared to the 70 kDa enzyme. An inhibition study revealed obvious differences between them. Thiol group inhibitors such as N-ethylmaleimide and leupeptin inhibited the 50 kDa enzyme but this was strongly activated by dithiothreitol, a thiol group stabilizer. Alternatively, the 70 kDa enzyme showed a sensitivity to a metal chelator and a serine group inhibitor indicating its metalloserine protease nature. The antifilarial drug diethylcarbamazine did not demonstrate any inhibition under in vitro conditions. Both enzymes were significantly inhibited by antibody IgG separated from Wuchereria bancrofti infected human sera, showing a possible immunoprotective role.     

Basidiomycetes harbour a hidden treasure of proteolytic diversity

This survey is the first to investigate the proteolytic potential of a large number of basidiomycetes. Aqueous extracts of 43 basidiomycetes were investigated for their content of proteolytic activities, using gelatin zymography. The activities were characterised qualitatively using class specific inhibitors. All four catalytic classes of proteases were present, with 4% of all activities classified as aspartic, 5% as cysteine, 6% as metallo and 22% as serine proteases, while the remaining activities could not be assigned unambiguously. The majority of the latter were not inhibited by any of the inhibitors used and were termed insensitive. Different proteolytic activities are evenly distributed among members of all orders of basidiomycetes, although some taxa are a richer source of proteases than others. A significant number of the cysteine protease activities shown here have not previously been reported in basidiomycetes. The fungal cysteine and serine protease inhibitors, clitocypin and CNSPI (Clitocybe nebularis serine protease inhibitor), both inhibited a number of activities and even a few activities that were otherwise insensitive to all other inhibitors used, hence indicating their potential for a regulatory role. The number and diversity of proteases in basidiomycetes are seen to be remarkable and encourage further investigation.     

Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98 degrees C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons [kDa]) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98 degrees C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% beta-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.     

Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus.

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98 degrees C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons [kDa]) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98 degrees C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% beta-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.     

Purification and characterization of a serine protease from Cucumis trigonus Roxburghi

Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family.     

Characterization of a membrane-associated serine protease in Escherichia coli.

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively (M. Pacaud, J. Bacteriol. 149:6-14, 1982). The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. We termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [3H]diisopropylfluorophosphate as an active-site labeling reagent, we determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, and endogenous, periplasmic inhibitor of trypsin.     

Characterization of excretory/secretory endopeptidase and metallo-aminopeptidases from Taenia crassiceps metacestodes

Cysticercosis is caused by Taenia spp. metacestodes, which must survive in the host tissues to complete their life cycle. Their survival depends on their control of host immune responses. Because many parasites use proteases to modulate host responses, we examined culture media from Taenia crassiceps metacestodes for protease activity using peptide substrates. We identified prominent aminopeptidase activity at neutral pH, which was inhibited by chelating agents and partially inhibited by the aminopeptidase inhibitor, bestatin. Endopeptidase substrates were optimally cleaved at slightly acidic pH and endopeptidase activity was inhibited by cysteine protease inhibitors. Gel filtration FPLC and subsequent visualization by silver staining revealed a metallo-aminopeptidase of molecular weight 21 kDa and cysteine proteases of Mr 70 and 64 kDA. Recombinant IL-2 was digested when incubated with parasite culture supernatants, but not with control media. IL-2 degradation was completely inhibited by 1,10 phenanthroline and partially inhibited by bestatin, suggesting that a metallo-aminopeptidase was responsible. Incubation of human IgG with culture supernatants resulted in complete degradation of IgG, which was blocked by cysteine protease inhibitors. These observations demonstrate that Taenia spp. metacestodes secrete a number of proteolytic enzymes, which may target molecules from the host immune system and assist in evasion of the host immune response.     

Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites

Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.     

Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.     

Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.     

Peptidohydrolases of Soybean Root Nodules : IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-l-Leu-beta-naphthylamide, while the second hydrolyzed n-benzoyl-l-Arg-beta-naphthylamine (Bz-l-Arg-betaNA), n-benzoyl-l-Arg-p-nitroanilide (Bz-l-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-l-Arg-betaNA, Bz-l-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-beta-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-beta-naphthylamides. The major activity of two of the aminopeptidases was directed toward l-Leu- and l-Met-beta-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-beta-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.     

Characteristic features in the structure and collagen-binding ability of a thermophilic collagenolytic protease from the thermophile Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in beta-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.