Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca²⁺ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca²⁺ ions and gramicidin S; its pH and temperature optima were 9.0 and 37°C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.     

In vitro fibrinolytic activity of an enzyme purified from Bacillus amyloliquefaciens strain KJ10 isolated from soybean paste

A fibrinolytic protease secreting producing Bacillus amyloliquefaciens strain KJ10 was initially screened from the fermented soybean. Maximum productivity was obtained in the culture medium after 40 h incubation, 34 °C incubation temperature at pH 8.0. Fibrinolytic protease production was enhanced in the culture medium with 1% sucrose (3712 ± 52 U/mL), 1% (w/v) yeast extract (3940 ± 28 U/mL) and 0.1% MgSO₄ (3687 ± 38 U/mL). Enzyme was purified up to 22.9-fold with 26%recovery after Q-Sepharose HP column chromatography. After three steps purification, enzyme activity was 1606U/mg and SDS-PAGE analysis revealed 29 kDa protein and enzyme band was detected by zymograpy. Enzyme was highly active at pH 8.0, at wide temperature ranges (40 °C ? 55 °C) and was activated by Mn²⁺ (102 ± 3.1%) and Mg²⁺ (101.4 ± 2.9%) ions. The purified fibrinolytic enzyme was highly specific against N-Suc-Ala-Ala-Pro-Phe-pNA (189 mmol/min/mL) and clot lytic activity reached 28 ± 1.8% within 60 minin vitro. The purified fibrinolytic enzyme showed least erythrocytic lysis activity confirmed safety to prevent various health risks, including hemolytic anemia. Based on this study, administration of fibrinolytic enzyme from B. amyloliquefaciens strain KJ10 is safe for clinical applications.     

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4 kDa, respectively. The enzymes were active at the temperature range of 50–60 °C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20 min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.     

A novel fibrinolytic serine protease from the polychaete Nereis (Neanthes) virens (Sars): purification and characterization

A novel fibrinolytic serine protease has been identified and purified to homogeneity from the coelomic fluid of polychaete Nereis (Neanthes) virens (Sars), and named N-V protease. N-V protease is a 29kDa single chain protein with an isoelectric point of pH 4.5. It hydrolyzes Aalpha-chain of fibrinogen with a high efficiency, and the Bbeta- and gamma-chains (Aalpha>Bbeta>gamma) with a lower efficiency. The proteolytic activity peaks at pH 7.8 is 45 degrees C. The activity is completely inhibited by serine protease inhibitors DFP (I(50)=5.8 x 10(-4)M) and PMSF (I(50)=5.5 x 10(-2)M), and almost completely by TLCK (I(50)=7.7 x 10(-1) M). But aprotinin, elastinal, SBTI, benzamidine, PCMB, EDTA, EGTA, iodoacetate, E64, and beta-mercaptoethanol have no effect on the protease activity. Therefore, N-V protease is identified as a serine protease. The primary amino acid sequence of N-V protease was determined by mass spectrometry (N-V protease, No. P83433). According to the MALDI-TOF MS analysis, there is no existing protein in the NCBI Non-redundant Protein Sequence Database that matches the N-V protease sequence. Therefore, N-V protease is a novel and special protein in N. virens. Furthermore, we have successfully established an expression cDNA library from the whole body of N. virens (data not shown).     

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.     

The 0.93A crystal structure of sphericase: a calcium-loaded serine protease from Bacillus sphaericus

We have previously isolated sphericase (Sph), an extracellular mesophilic serine protease produced by Bacillus sphaericus. The Sph amino acid sequence is highly homologous to two cold-adapted subtilisins from Antarctic bacilli S39 and S41 (76% and 74% identity, respectively). Sph is calcium-dependent, 310 amino acid residues long and has optimal activity at pH 10.0. S41 and S39 have not as yet been structurally analysed.In the present work, we determined the crystal structure of Sph by the Eu/multiwavelength anomalous diffraction method. The structure was extended to 0.93A resolution and refined to a crystallographic R-factor of 9.7%. The final model included all 310 amino acid residues, one disulfide bond, 679 water molecules and five calcium ions. Although Sph is a mesophilic subtilisin, its amino acid sequence is similar to that of the psychrophilic subtilisins, which suggests that the crystal structure of these subtilisins is very similar.The presence of five calcium ions bound to a subtilisin molecule, as found here for Sph, has not been reported for the subtilisin superfamily. None of these calcium-binding sites correlates with the well-known high-affinity calcium-binding site (site I or site A), and only one site has been described previously. This calcium-binding pattern suggests that a reduction in the flexibility of the surface loops of Sph by calcium binding may be responsible for its adaptation to mesophilic organisms.     

Structure-activity studies of cyclic ketone inhibitors of the serine protease plasmin: design, synthesis, and biological activity

Three series of cyclic ketone inhibitors were synthesized and evaluated against the serine protease plasmin. Peptide inhibitors that incorporated 3-oxotetrahydrofuran and 3-oxotetrahydrothiophene 1,1-dioxide groups had the highest activities. Alkylamino substituents, which were designed to bind in the S1 subsite of plasmin, were attached to the inhibitors. Compounds 5c and 5g, which incorporated 6-aminohexyl substituents, were found to be optimal and demonstrated IC₅₀ values in the low micromolar range. Incorporating conformationally constrained peptide segments into the inhibitors did not improve their activities.     

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

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.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium,Francisella tularensis

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.     

一株产纤溶酶菌株的分离鉴定及其纤溶组分分析

【目的】筛选性能良好的产纤溶酶菌株,对菌株进行多项分类鉴定,分析其纤溶酶系的组成特征及纤溶能力。【方法】通过酪蛋白培养基初筛,琼脂-纤维蛋白双层平板复筛,从海泥、土壤等环境中筛选纤维蛋白降解菌,以尿激酶为标准测定纤溶酶活性。通过形态学、生理生化特征研究,结合16S rDNA基因序列分析菌株种类及系统分类地位。通过SDS-PAGE和纤维蛋白酶谱法分析胞外纤溶酶系的组成特征。【结果】筛选到一株能降解纤维蛋白的细菌CNY16,鉴定其为沙福芽孢杆菌(Bacillus safensis)。该酶为胞外酶,SDS-PAGE和纤维蛋白酶谱结果表明该纤溶酶系有至少两种分子量大小不同的纤溶酶,分别约33 kD和23 kD。能有效溶解血块中纤维蛋白,并且对红细胞无降解作用。【结论】细菌CNY16是一株新的纤溶酶产生菌,纤溶酶活性及稳定性较好,具有潜在开发价值。为获取新型纤溶酶提供了一种新的菌源。     

Isolation and characterization of a novel serine protease inhibitor,SjSPI, from Schistosoma japonicum

Serine proteinase inhibitor (Serpin, SPI) is a vital superfamily of endogenous inhibitors that monitor proteolytic events active in a number of biological functions. In this study, we isolated a full length gene encoding a novel serine protease inhibitor of Schistosoma japonicum (SjSPI) and characterized its molecular properties. Our result showed that SjSPI contained an open reading frame of 1,218?bp, which encoded 405 amino acid residues. Chromosomal structure analysis showed that SjSPI gene was comprised of six exons separated by five introns. It had essential structural motifs which were well conserved among the Serpin superfamily and showed 17-33% sequence identities with Serpins from other helminthic parasites. Trematode Serpin diverged separately into two different subclades and that the SjSPI clustered Subclade I. Exon-intron structures of trematode Serpins were highly conserved, closely with cestode Serpins. No signal peptide but a strongly transmembrane domain was predicted to exist in SjSPI, suggesting that the protein might be a soluble membrane-associated protein. Homology modeling predicted in silico confirmed that the SjSPI structure also belonged to the Serpin superfamily, containing nine α-helices and a reactive central loop. The bacterially expressed recombinant GST-SjSPI protein effectively inhibited the activities of chymotrypsin, trypsin and thrombin. Expression of SjSPI was detected throughout various developmental stages of the parasite in host and reached its maximal levels at the adult and egg stages, which suggests that SjSPI may be possibly involved in maintaining the physiology of eggs by regulating endogenous serine proteases.     

Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.