| thermostability at ultrahigh temperatures of thermolysin and a protease from a psychrotrophic pseudomonas. | thermal inactivation at 110-150 degrees c of thermolysin (ec 3.4.24.4), produced by the thermophile bacillus thermoproteolyticus, and the extracellular protease of pseudomonas sp. mc60 a psychotroph, were investigated at 130 degrees c, both enzymes had approximately the same deltah (22 kcal/mol) and deltas (-13.5 cal/mol per degree) values. both enzymes contain zinc and calcium. the amino acid compositions of the enzymes were similar except that mc60 protease exhibited a more typical tyrosine co ... | 1977 | 411519 |
| effect of glu-143 and his-231 substitutions on the catalytic activity and secretion of bacillus subtilis neutral protease. | on the basis of the homology with the bacillus thermoproteolyticus zinc endopeptidase thermolysin, we hypothesized that glu-143 and his-231 are the key residues for the catalytic activity of the bacillus subtilis neutral protease. to test this possibility by site-directed mutagenesis, we substituted these two residues with ala, ser, trp and arg, and leu, val and cys respectively. all these substitutions dramatically affected the amount of secreted mutant proteins, as determined by immunological ... | 1989 | 2494652 |
| studies on the bacillus subtilis neutral-protease- and bacillus thermoproteolyticus thermolysin-catalyzed hydrolysis of dipeptide substrates. | | 1970 | 4989948 |
| [characteristics of a new bioindicator and its testing in practice assays of thermal disinfection methods]. | thermolysin (ec 3.4.24.4) is a thermostable metalloproteinase isolated from bacillus thermoproteolyticus. it has been tested for its suitability as a macromolecular bioindicator for the screening of thermal disinfection methods. using the time- und temperature-dependent inactivation rate of the immobilized enzyme we get a measure if given parameters of disinfection have been maintained. in order to determine standard conditions of practical use, physico-chemical properties of the enzyme (ph- and ... | 1990 | 2261056 |
| characterization of an extracellular metalloprotease with elastase activity from staphylococcus epidermidis. | the gene sepa from staphylococcus epidermidis tu3298-p, encoding the extracellular neutral metalloprotease sepp1, was cloned into pt181mcs. dna sequencing revealed an open reading frame of 1,521 nucleotides encoding a 507-amino-acid protein with an m(r) of 55,819. the sepa-containing dna fragment did not hybridize with staphylococcus hyicus or staphylococcus carnosus dna. expression of sepa in the protease-negative s. carnosus (pt181mcsp1) resulted in overproduction of a 33-kda protease found in ... | 1993 | 8320236 |
| erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene. | the prt1 gene encoding extracellular protease from erwinia carotovora subsp. carotovora ec14 in cosmid pca7 was subcloned to create plasmid psk1. the partial nucleotide sequence of the insert in psk1 (1,878 bp) revealed a 1,041-bp open reading frame (orf1) that correlated with protease activity in deletion mutants. orf1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 da. escherichia coli transformed with psk1 or psk23, a subclone of psk1, produces a protease ( ... | 1991 | 1917878 |
| cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of vibrio proteolyticus. | the gene (nprv), encoding the extracellular neutral protease, vibriolysin (nprv), of the gram- marine microorganism, vibrio proteolyticus, was isolated from a v. proteolyticus dna library constructed in escherichia coli. the recombinant e. coli produced a protease that co-migrated with purified neutral protease from v. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate n-[3-(2-furyl)acryloyl]-l-alanylphenylalani ... | 1992 | 1551587 |
| structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis. | crystal structures are known for three members of the bacterial neutral protease family: thermolysin from bacillus thermoproteolyticus (tln), the neutral protease from bacillus cereus (neu), and the elastase of pseudomonas aeruginosa (pae), both in free and ligand-bound forms. each enzyme consists of an n-terminal and c-terminal domain with the active site formed at the junction of the two domains. comparison of the different molecules reveals that the structure within each domain is well conser ... | 1992 | 1445869 |
| the role of bound calcium ions in thermostable, proteolytic enzymes. ii. studies on thermolysin, the thermostable protease from bacillus thermoproteolyticus. | the functional properties of the four calcium ions, bound by thermolysin, appear to be very similar to those of the single calcium ion bound by thermomycolase (g. voordouw and r.s. roche (1975), biochemistry, preceding paper in this issue). hence when the free calcium ion concentration is varied in the range where the calcium double-site dissociates (g. voordouw and r.s. roche (1974), biochemistry 13, 5017), no changes are observed in the sedimentation coefficient or the peptide circular dichroi ... | 1975 | 1182109 |
| thermal stability of homologous neutral metalloendopeptidases in thermophilic and mesophilic bacteria: structural considerations. | thermolysin and neutral protease a are neutral metalloendopeptidases having similar specificity, molecular weight, metal content, and amino acid composition. thermolysin, derived from the thermophilic organism bacillus thermoproteolyticus, is heat inactivated at about 84 degrees whereas neutral protease a, derived from the mesophilic organism bacillus subtilis, is inactivated at about 59 degrees. structural analyses reveal that the two enzymes are homologous. of the 326 residues of neutral prote ... | 1976 | 820564 |
| evidence of homologous relationship between thermolysin and neutral protease a of bacillus subtilis. | a comparison of the partial amino-acid sequence of neutral protease a from bacillus subtilis with the structure of thermolysin (ec 3.4.24.4) from bacillus thermoproteolyticus reveals that these two proteins are homologous. of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include nine of the 13 residues previously identified as components of the active site of thermolysin. this similarity provides support for the ... | 1975 | 812093 |
| deviations from compositional randomness in eukaryotic and prokaryotic proteins: the hypothesis of selective-stochastic stability and a principle of charge conservation. | eight proteins of diverse lengths, functions, and origin, are examined for compositional non-randomness amino acid by amino acid. the proteins investigated are human fibrinopeptide a, guinea pig insulin, rattlesnake cytochrome c, ms2 phage coat protein, rabbit triosephosphate isomerase, bovine pancreatic deoxyribonuclease a, bovine glutamate dehydrogenase, and bacillus thermoproteolyticus thermolysin. as a result of this study the experimentally testable hypothesis is put forth that for a large ... | 1975 | 173858 |
| cloning and expression in bacillus subtilis of the npr gene from bacillus thermoproteolyticus rokko coding for the thermostable metalloprotease thermolysin. | we report the isolation, cloning and expression, in bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by bacillus thermoproteolyticus rokko. the nucleotide sequence has revealed that, like neutral proteases produced by other members of the bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical n-terminal membrane signal sequence. further, the thermolysin gene shares a strong homology with two other previously ... | 1994 | 8002967 |
| crystal structure of the ferredoxin i from desulfovibrio africanus at 2.3 a resolution. | the crystal structure of the ferredoxin i from the sulfate-reducing bacterium desulfovibrio africanus (dafdi) has been solved and refined by x-ray diffraction. the crystals are orthorhombic with a = 96.6 a, b = 58.1 a, and c = 20.7 a, space group p2(1)2(1)2, and two ferredoxin molecules per asymmetric unit. the initial electron density map has been obtained by combining phasing by molecular replacement methods, anomalous scattering, and noncrystallographic averaging. the final crystallographic r ... | 1994 | 7803404 |
| structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin. | tetanus neurotoxin (tent) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(vamp)/synaptobrevin. tent is the prototype of clostridial neurotoxins, a new family of metalloproteinases. they consist of three domains and the proteolytic activity is displayed by the 50-kda light chain (l chain). the l chain was isolated here in the native state from bacterial filtrates of clostridium tetani and its structure was studied via circular dich ... | 1995 | 7744050 |
| atomic resolution structures of oxidized [4fe-4s] ferredoxin from bacillus thermoproteolyticus in two crystal forms: systematic distortion of [4fe-4s] cluster in the protein. | diffraction data of two crystal forms (forms i and ii) of [4fe-4s] ferredoxin from bacillus thermoproteolyticus have been collected to 0.92 a and 1.00 a resolutions, respectively, at 100 k using synchrotron radiation. anisotropic temperature factors were introduced for all non-hydrogen atoms in the refinement with shelx-97, in which stereochemical restraints were applied to the protein chain but not to the [4fe-4s] cluster. the final crystallographic r-factors are 9.8 % for 7.0-0.92 a resolution ... | 2002 | 11827483 |
| preliminary x-ray diffraction studies on a [4fe-4s] ferredoxin from bacillus thermoproteolyticus. | | 1981 | 7334523 |
| thermolysin and bacillus subtilis neutral protease. conformation and stability of two homologous neutral metalloendopeptidases. | a comparative study on thermolysin from bacillus thermoproteolyticus and neutral protease from bacillus subtilis involving (far- and near-ultraviolet circular dichroism (cd) and immunological techniques is reported. these enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. the far-ultraviolet cd spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of elliptic ... | 1980 | 6780484 |
| pressure dependence of thermolysin catalysis. | a comparison of the pressure and temperature dependences of the catalytic reaction of thermolysin, a thermostable neutral protease from bacillus thermoproteolyticus, with those of a non-thermostable neutral protease from bacillus subtilis revealed a distinct difference in km values of these enzymes for 3-(2-furyl)acrylyl-blocked dipeptide and tripeptide substrates, but not for kcat. namely, the volume changes for the binding process (delta v) for these substrates and several competitive inhibito ... | 1984 | 6432533 |
| classification of iron-sulfur cores in ferredoxins by 1h nuclear magnetic resonance spectroscopy. | a 1h nuclear magnetic resonance (nmr) study was carried out on various ferredoxins which possess one of three types of iron-sulfur clusters, (2fe-2s), (3fe-3s), or (4fe-4s). in the isolated form, (2fe-2s) ferredoxins from spinach (spinacea oleracia), pokeweed (phytolacca americana), a blue-green alga (spirulina platensis), and a halobacterium (halobacterium halobium) exhibited two broad resonances common in chemical shift at the region downfield of 10 ppm. in their reduced forms, seven contact-s ... | 1983 | 6417123 |
| inhibition of metalloendopeptidases by 2-mercaptoacetyl-dipeptides. | a series of 2-mercaptoacetyl-dipeptides, a potential group of metalloendopeptidase inhibitors, has been synthesized by coupling the n-hydroxysuccinimide ester of s-acetyl-2-mercaptoacetic acid with hydrophobic dipeptide methyl ester hydrochlorides, followed by hydrolysis with naoh in aqueous methanol and acidification with hcl. thus, the 2-mercaptoacetyl derivatives of l-phenylalanyl-l-leucine, l-leucyl-l-phenylalanine and l-leucyl-d-phenylalanine were prepared. the first two compounds inhibit e ... | 1983 | 6413206 |
| the interaction of alpha2-macroglobulin with proteinases. binding and inhibition of mammalian collagenases and other metal proteinases. | 1. experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. a specific collagenase from rabbit synovial cells was inhibited by human serum. the inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-macroglobulin presaturated with trypsin or cathepsin b1 did not inhibit collagenase, and pretr ... | 1974 | 4374931 |
| differential scanning calorimetry of the irreversible thermal denaturation of thermolysin. | a differential scanning calorimetry study of the thermal denaturation of bacillus thermoproteolyticus rokko thermolysin was carried out. the calorimetric traces were found to be irreversible and highly scan-rate dependent. the shape of the thermograms, as well as their scan-rate dependence, can be explained by assuming that the thermal denaturation takes place according to the kinetic scheme n k----d, where k is a first-order kinetic constant that changes with temperature, as given by the arrhen ... | 1988 | 3365417 |
| tertiary structure of bacillus thermoproteolyticus [4fe-4s] ferredoxin. evolutionary implications for bacterial ferredoxins. | the structure of a low-potential [4fe-4s] ferredoxin from bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic r-factor of 0.33, with 2.3 a (1 a = 0.1 nm) resolution data. the least-squares refinement based on the bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 a, apart. the nh ... s hydrogen bonds betwe ... | 1988 | 3351918 |
| cloning and nucleotide sequence of the highly thermostable neutral protease gene from bacillus stearothermophilus. | the gene (nprm) for the highly thermostable neutral protease of bacillus stearothermophilus mk232 was cloned in bacillus subtilis using ptb53 as a vector. the nucleotide sequence of nprm and its flanking regions was determined. the dna sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. a shine-dalgarno (sd) sequence was found 12 bases upstream from the translation start site (atg). a possible promotor sequence (ttttcc for the -35 region ... | 1988 | 3149972 |
| structure and stability of thermophilic enzymes. studies on thermolysin. | the molecular mechanisms responsible for the unusual stability of enzymes isolated from thermophilic microorganisms are much more complex and subtle than was originally thought. in particular, a general mechanism cannot be proposed, since individual enzymes can be stabilized by specific molecular interactions and forces. the results of studies on thermophilic enzymes obtained in recent years in our laboratory will be summarized, with particular emphasis being placed on those obtained with thermo ... | 1988 | 3129040 |
| nucleotide sequence and promoter region for the neutral protease gene from bacillus stearothermophilus. | the thermostable neutral protease gene nprt of bacillus stearothermophilus was sequenced. the dna sequence revealed only one large open reading frame, composed of 1,644 bases and 548 amino acid residues. a shine-dalgarno sequence was found 9 bases upstream from the translation start site (atg), and the deduced amino acid sequence contained a signal sequence in its amino-terminal region. the sequence of the first 14 amino acids of purified extracellular protease completely matched that deduced fr ... | 1985 | 2993245 |
| virulence of metalloproteases produced by vibrio species on pacific oyster crassostrea gigas larvae. | vibrio tubiashii, a pathogen of shellfish larvae and juveniles, produces several extracellular products. here, we document that culture supernatants of several marine vibrio species showed toxicity to oyster larvae. treatment of these supernatants with edta not only severely diminished proteolytic activities, but also dramatically reduced toxicity to the larvae. culture supernatants of metalloprotease-deficient mutants of v. tubiashii, v. cholerae, and v. splendidus were impaired in their abilit ... | 2009 | 19694172 |
| molecular characterization and nucleotide sequence of the pseudomonas aeruginosa elastase structural gene. | the elastase structural gene (lasb) from pseudomonas aeruginosa pao1 has been previously cloned on an 8-kilobase (kb) dna fragment. the lasb gene, cloned in both orientations in puc18, produced elastase in escherichia coli, indicating that its promoter and translation initiation sites are functional in e. coli. deletion analysis further defined the location of the lasb gene to a 3.0-kb ecori-kpni fragment (prb1803). elastase prepared from e. coli tb1 (prb1803) corresponded in molecular weight to ... | 1988 | 2842313 |
| structure of [4fe-4s] ferredoxin from bacillus thermoproteolyticus refined at 2.3 a resolution. structural comparisons of bacterial ferredoxins. | the structure of a low-potential ferredoxin isolated from bacillus thermoproteolyticus has been refined by a restrained least-squares method. the final crystallographic r factor is 0.204 for 2906 reflections with f greater than 3 sigma f in the 6.0 to 2.3 a resolution range. the model contains 81 amino acid residues, one [4fe-4s] cluster, and 59 water molecules. the root-mean-square deviation from ideal values for bond lengths is 0.018 a, and the mean coordinate error is estimated to be 0.25 a. ... | 1989 | 2600971 |
| [renaturation of bacterial metalloproteinases]. | after denaturation by phenol or acid ethanol bacterial metalloproteinases secreted by bacillus thermoproteolyticus and bacillus megaterium cells can be renatured by dissolution in 99.7% formic acid with subsequent dilution of the solution and its neutralization with an appropriate amount of an alkali. renaturation is optimal at ph 9.0 in the presence of 30% glycerol as stabilizer, ca2+ and zn2+ ions needed for the formation of a native structure and reconstitution of the enzyme catalytic center. ... | 1993 | 8364114 |
| site-directed mutagenesis of glu-141 and his-223 in pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against pseudomonas infection. | both pseudomonas aeruginosa elastase and bacillus thermoproteolyticus thermolysin are zinc metalloproteases. on the basis of the high homology of the p. aeruginosa elastase with the bacillus thermolysin, we hypothesized that glu-141 and his-223 are the key residues for catalytic activity of the pseudomonas elastase. to test this possibility, we replaced glu-141 with asp, gln, and gly and his-223 with gly, glu, and leu by site-directed mutagenesis. these substitutions dramatically diminished the ... | 1993 | 8454342 |
| chemotaxis of alveolar macrophages and neutrophils in response to microbial products derived from organic dust. | mechanisms of chemotaxis of alveolar macrophages (ams) and neutrophils (pmns) in response to microbial products derived from organic dust were studied using the blindwell chemotaxis chamber technique. seven different known etiological agents causing respiratory symptoms were used for experiments: cell extract and endotoxin from pantoea agglomerans (synonyms: erwinia herbicola, enterobacter agglomerans), cell extracts from thermoactinomyces vulgaris and aspergillus fumigatus, protease from bacill ... | 1995 | 8705013 |
| the roles of the prosequence of thermolysin in enzyme inhibition and folding in vitro. | the zinc endopeptidase thermolysin (ec 3.4.24.27), an extracellular enzyme from bacillus thermoproteolyticus, is synthesized as a preproprotein, with the prosequence (204 residues) being two-thirds the size of the mature enzyme (316 residues). this prosequence, expressed in and purified from escherichia coli, inhibited thermolysin in vitro with an ic50 value of 14 nm. it also inhibited a closely related enzyme produced by bacillus stearothermophillus, albeit with a 16-fold higher ic50 value (220 ... | 1996 | 8900115 |
| model building of a thermolysin-like protease by mutagenesis. | the present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of bacillus subtilis (np-sub). an initial model of np-sub was constructed using the crystal structures of the homologous neutral proteases of bacillus thermoproteolyticus (thermolysin) and bacillus cereus as templates. the largest portion of np-sub could be modelled satisfactorily, using standard techniques, but several surface-located regions could only be mo ... | 1997 | 9153087 |
| the prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in escherichia coli. | the zinc metalloendopeptidase, thermolysin (ec 3.4.24.27) produced by bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme. thermolysin is synthesised as a pre-proenzyme, with an n-terminal prosequence of 204 residues and a mature sequence of 316 residues. the prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the ... | 1999 | 9925774 |
| activity and stability of a neutral protease from vibrio sp. (vimelysin) in a pressure-temperature gradient. | the apparent second-order rate constant of hydrolysis of fua-gly-leunh2 by vimelysin, a neutral protease from vibrio sp. t1800, was measured in a variable pressure-temperature gradient (0. 1-400 mpa and 5-40 degrees c). the apparent maximum rate was observed at approximately 15 degrees c and 150-200 mpa; the pressure-activation ratio (kcat/km(max)/kcat/km(0.1 mpa)) was reached about sevenfold. the pressure dependence of the kcat and km parameters at constant temperature (25 degrees c) revealed t ... | 2000 | 10672005 |
| the 2.2 a resolution structure of thermolysin (tln) crystallized in the presence of potassium thiocyanate. | a new crystallization protocol for thermolysin (ec 3.4.24.27) from bacillus thermoproteolyticus is presented. after dissolving the protein in the presence of kscn, which avoids the use of dmso and cscl, crystals were obtained following the salting-in method. crystal cell parameters are isomorphous with those previously reported from dmso/cscl mixtures. the new scn(-) crystal structure has been analyzed. it shows the presence of one thiocyanate ion in the catalytic site and several rearrangements ... | 2002 | 12454500 |
| gene cloning and characterization of a bacillus vietnamensis metalloprotease. | a bacillus vietnamensis metalloprotease (bvmp) with high affinity toward collagen was isolated and purified from the culture supernatant of bacillus vietnamensis 11-4 occurring in vietnamese fish sauces. the bvmp gene was cloned and its nucleotide and coded amino acid sequences determined. bvmp consists of 547 amino acid residues, with the zinc-binding sites conserved in common metalloproteases. it shares 57% amino acid identity with thermolysin originating from bacillus thermoproteolyticus. the ... | 2004 | 15277758 |
| protein control of iron-sulfur cluster redox potentials. | the relationship between the three-dimensional structures of iron-sulfur proteins and the redox potentials of their iron-sulfur clusters is of fundamental importance. we report calculations of the redox potentials of the [fe4s4(s-cys)4]-2/-3 couple in four crystallographically characterized proteins: azotobacter vinelandii ferredoxin i, peptococcus aerogenes ferredoxin, bacillus thermoproteolyticus ferredoxin, and chromatium vinosum high potential iron protein (hipip). our calculations use the " ... | 1992 | 1464583 |
| purification, characterization cloning, and sequencing of metalloendopeptidase from streptomyces septatus th-2. | streptomyces septatus th-2 secretes a large amount of a protease when cultured on a medium containing k(2)hpo(4) and glucose. the enzyme was purified to homogeneity by a three-step procedure. this enzyme had a molecular mass of approximately 35kda, and was particularly inhibited by edta and phosphoramidon. its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries. the protease was found to prefer phe and tyr at the p(1) position, a hydrophobic or ... | 2005 | 15639229 |
| the protein-protein interactions between smpi and thermolysin studied by molecular dynamics and mm/pbsa calculations. | thermolysin is a zinc-metalloendopeptidase secreted by the gram-positive thermophilic bacterium bacillus thermoproteolyticus. thermolysin belongs to the gluzinicin family of enzymes, which is selectively inhibited by steptomyces metalloproteinase inhibitor (smpi). very little is known about the interaction between smpi and thermolysin. knowledge about the protein-protein interactions is very important for designing new thermolysin inhibitors with possible industrial or pharmaceutical application ... | 2005 | 15702924 |
| functional analysis of the burkholderia cenocepacia zmpa metalloprotease. | burkholderia cenocepacia zmpa is expressed as a preproenzyme typical of thermolysin-like proteases such as pseudomonas aeruginosa lasb and bacillus thermoproteolyticus thermolysin. the zmpa gene was expressed using the ppro-exhta his(6) tag expression system, which incorporates a six-his tag at the n-terminal end of the protein, and recombinant zmpa was purified using ni-nitrilotriacetic acid affinity chromatography. upon refolding of the recombinant his(6)-pre-pro-zmpa (62 kda), the fusion prot ... | 2005 | 15968051 |
| identification of variant molecules of bacillus thermoproteolyticus ferredoxin: crystal structure reveals bound coenzyme a and an unexpected [3fe-4s] cluster associated with a canonical [4fe-4s] ligand motif. | during the purification of recombinant bacillus thermoproteolyticus ferredoxin (btfd) from escherichia coli, we have noted that some fe-s proteins were produced in relatively small amounts compared to the originally identified btfd carrying a [4fe-4s] cluster. these variants could be purified into three fe-s protein components (designated as v-i, v-ii, and v-iii) by standard chromatography procedures. uv-vis and epr spectroscopic analyses indicated that each of these variants accommodates a [3fe ... | 2005 | 16156653 |
| extracellular production of recombinant thermolysin expressed in escherichia coli, and its purification and enzymatic characterization. | thermolysin is a representative zinc metalloproteinase derived from bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. extracellular production of thermolysin has been achieved in bacillus, but not in escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. in this study, we expressed thermolysin as a single polypeptide pre-proenzyme in e. coli under the original promoter s ... | 2006 | 16169746 |
| molecular cloning of the gene encoding vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents. | vimelysin is a unique metalloproteinase from vibrio sp. t1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. a 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an n-terminal pro-region, a mature enzyme, and a c-terminal pro-region. the mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from v. vulnificus, pseudolysin from pseudomonas aeruginosa, and ... | 2005 | 16428299 |
| a new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in escherichia coli. | thermolysin, a representative zinc metalloproteinase from bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. the conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. in this study, we have established a new expression method that does not require ... | 2007 | 17616558 |
| engineering, expression, purification, and production of recombinant thermolysin. | thermolysin [ec 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of bacillus thermoproteolyticus rokko. since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. thermolysin is used in industry, esp ... | 2007 | 17875473 |
| origins of the different metal preferences of escherichia coli peptide deformylase and bacillus thermoproteolyticus thermolysin: a comparative quantum mechanical/molecular mechanical study. | the escherichia coli peptide deformylase (pdf) and bacillus thermoproteolyticus thermolysin (tln) are two representative metal-requiring peptidases having remarkably similar active centers but distinctively different metal preferences. zinc is a competent catalytic cofactor for tln but not for pdf. reaction pathways and the associated energetics for both enzymes were determined using combined semiempirical and ab initio quantum mechanical/molecular mechanical modeling, without presuming reaction ... | 2008 | 18651766 |
| determinants for psychrophilic and thermophilic features of metallopeptidases of the m4 family. | naturally occurring peptidases from organisms living under extreme conditions are adapted to function in environmental extremes, including temperature, salinity, ph, or pressure. these organisms represent unique sources for new bio-molecules that have both industrial and medicinal application. adaptive strategies for functioning under extreme conditions are reflected at the enzyme sequence and structural level. understanding the determinants responsible for unique functional features can be used ... | 2009 | 19795569 |
| synergistic effect of neutral protease and clostripain on rat pancreatic islet isolation. | islet isolation currently requires collagenase, neutral protease and other components. thermolysin (tl) from bacillus thermoproteolyticus is the gold standard neutral protease. however, we speculated that neutral protease derived from clostridium histolyticum (ch; chnp) would be biologically superior for islet isolation. tryptic-like activity has also been reported to be important. therefore, we focused on clostripain (cp), since it is one of the main proteases in clostridium histolyticum which ... | 2015 | 25803499 |
| a new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. | the optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. in this study, we evaluated eight different enzyme combinations (ecs) in an attempt to improve islet yield. the ecs consisted of purified, intact or truncated class 1 (c1) and class 2 (c2) collagenases from clostridium histolyticum (ch), and neutral protease (np) from bacillus thermoproteolyticus rokko (thermolysin) or ch (chnp). | 2012 | 22318245 |
| mutational analysis of potential zinc-binding residues in the active site of the enterococcal d-ala-d-ala dipeptidase vanx. | vanx, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic enterococci, is a zinc-containing d-ala-d-ala dipeptidase. to identify potential zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue vanx protein. of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating his116, asp123, an ... | 1997 | 9265630 |
| intramolecular processing of prothermolysin. | thermolysin, an extracellular zinc endopeptidase from bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (o'donohue, m. j., and beaumont, a. (1996) j. biol. chem. 271, 26477-26481). to determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, n-terminal histidine-tagged pr ... | 1998 | 9488701 |
| catalytic activity of thermolysin under extremes of pressure and temperature: modulation by metal ions. | the catalytic activity of thermolysin (tl), a zn-dependent neutral protease from bacillus thermoproteolyticus, has been studied over a wide interval of pressures (1 bar to 4 kbar) and temperatures (20 degreesc to 80 degreesc) by monitoring hydrolysis of a low-molecular-mass substrate, 3-(2-furylacryloyl)-glycyl-l-leucine amide. this reaction shows a very large negative value for the activation volume and, because of that, simultaneous increase in temperature and pressure leads to a significant ( ... | 1998 | 9675281 |
| amino-acid sequence and three-dimensional structure of the staphylococcus aureus metalloproteinase at 1.72 a resolution. | aureolysin is an extracellular zinc-dependent metalloproteinase from the pathogenic bacterium staphylococcus aureus. this enzyme exhibits in vitro activity against several molecules of biological significance for the host, indicating that it is involved in the pathology of staphylococcal diseases. | 1998 | 9753696 |
| vibrio cholerae hemagglutinin(ha)/protease: an extracellular metalloprotease with multiple pathogenic activities. | vibrio cholerae of serogroup o1 and o139, the etiological agent of the diarrheal disease cholera, expresses the extracellular zn-dependent metalloprotease hemagglutinin (ha)/protease also reported as vibriolysin. this enzyme is also produced by non-o1/o139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). orthologs of ha/protease are present in other members of the vibrionaceae family pathogenic to humans and fish. ha/protease belongs to the ... | 2016 | 26952544 |
| enzymatically triggered peptide hydrogels for 3d cell encapsulation and culture. | we have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. based on recent work done on the enzymatically triggered gelation of fefk (f, phenylalanine; e, glutamic acid; k, lysine) using thermolysin, a protease enzyme from bacillus thermoproteolyticus rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3d hydrogel matrix. first, the properties of enzymaticall ... | 2014 | 24920105 |
| effect of enzyme concentration of the morphology and properties of enzymatically triggered peptide hydrogels. | we have recently shown that thermolysine, a protease enzyme obtained from bacillus thermoproteolyticus rokko , can be used to trigger the gelation of fefk (f, phenylalanine; e, glutamic acid; k, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. in this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using hplc, ftir, re ... | 2013 | 23506527 |
| purification and characterization of a thermolysin like protease from thermoactinomyces thalpophilus mcmb-380. | the extracellular thermolysin like protease (tlp) was purified and characterized from thermoactinomyces thalpophilus mcmb-380 (genbank accession no. ef397000). the enzyme was purified to homogeneity by successive ultra filtration steps using 50 kda and 10 kda membrane filters followed by anion exchange chromatography. the molecular mass and isoelectric point of the enzyme were found to be 34.4 kda and 9.5, respectively. the proteolytic activity was inhibited by edta and the enzyme required ca2+ ... | 2013 | 23360323 |