Purification and Characterization of a Thermostable Neutral Metalloprotease I from Chloroflexus aurantiacus J-10-fl

Chloroflexus aurantiacus J-10-fl was found to contain two types (protease I and protease II) of thermostable proteases which were separated by Butyl-Toyopearl 650M chromatography. Protease I was purified to electrophoretic homogeneity from the culture broth of C. aurantiacus J-10-fl. The molecular mass of protease I was estimated to be approximately 66 kDa by SDS-PAGE, and the value of approximately 66kDa was also obtained by the Hedrick-Smith method, indicating that protease I was a monomer. The isoelectric point was 6.2. Protease I activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, and o-phenanthroline. The optimum pH for the activity of protease I was around 8.0. Addition of Ca²⁺ increased the pH and heat stabilities of protease I. The activity was stable between pH 4.0–11.0 and up to 75°C, and the maximum activity was observed at 70°C in the presence of 2mM CaCl₂. Protease I was resistant to the treatment by denaturing reagents (8 M urea or 1% SDS) at pH 8.0 and 20°C for 24 h. The sites of cleavage. in oxidized insulin B chain by protease I were similar to those by other microbial neutral metalloproteases. Elastase activity of protease I was not detected.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

T sujibo , H., M iyamoto , K., H asegawa , T. & I namori , Y. 1990. Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete. Journal of Applied Bacteriology 69 , 520–529.
Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21000 and 36000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10–12 (protease I) and pH 10.5 (protease II). The optimum temperature for the activity of protease I was 70°C and that for protease II was 60°C. Protease I was stable in the range of pH 4.0–8.0 up to 60°C and protease II was stable in the range of pH 6.0–12.0 up to 50°C.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21,000 and 36,000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10-12 (protease I) and pH 10.5 (protease II). The optimum temperture for the activity of protease I was 70 degrees C and that for protease II was 60 degrees C. Protease I was stable in the range of pH 4.0-8.0 up to 60 degrees C and protease II was stable in the range of pH 6.0-12.0 up to 50 degrees C.     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP intracellular serine protease - ISP-A-Bsu A-50 and ISP-B-Bsu A-50 molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively - SDS sodium dodecyl sulfate - PMSF phenylmethyl sulfonylfluoride - pNA p-nitroanilide - Buffer A 50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl₂ adjusted to pH 8.5 with HCl     

Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.     

Proteases of the genus Bacillus. I. Neutral proteases

B. subtilis NRRL B3411 neutral protease has been extensively purified by solvent, and salt fractional ion, pigment removal with DEAE-cellulose followed by chromatography on hydroxylapatite, and a final passage through a Sephadex G-100 column. The neutral protease was shown to be homogeneous by disc gel and cellulose acetate electrophoresis, gel filtration chromatography, and ultra-centrifugation. The molecular weight was determined by osmometry and ultracentrifugation to be about 38–42,000 and the amino acid composition and zinc content determined. The general properties of the enzyme, pH-activity relationship, stability, effect of inhibitors, and specificity are discussed. Comparative studies were carried out on the B. subtilis NRRL B3411 and B. subtilis var. amylosacchariticus neutral proteases and these enzymes were found to be indistinguishable by the methods used, but quite distinct from the thermostable enzyme thermolysin from B. thermoprotcolyticus.     

Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose

Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5–8 and 37–45 °C when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7–8 and at temperature close to 35°C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40–45°C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.     

Physiological and nutritional determinants of protease secretion by Clostridium sporogenes: characterization of six extracellular proteases

Summary Proteases were the principal secretory proteins of Clostridium sporogenes and were optimally produced after active growth at 37° C. Glucose, ammonia and peptides repressed protease production. Protease formation was maximal in cultures grown at pH 6.5, but proteolytic activity exhibited a pH optimum of 7.0–8.0. Protease activity in culture filtrates was stimulated by divalent metal ions (Ca²⁺, Mn²⁺ and Co²⁺) and was strongly inhibited by ethylene diaminetetraacetate (EDTA) and thimerosal. Non-denaturing polyacrylamide gel electrophoresis and HPLC gel filtration demonstrated the presence of six major proteases of low molecular mass (approx. 15–35 kDa). The enzymes were partially purified from non-denaturing gels. Each hydrolysed azocoll and azocasein, but differed in their activity against a range of native collagen substrates. All six enzymes degraded human placental collagen (Type IV) but only one had a broad substrate specificity, being able to hydrolyse the more recalcitrant collagens (Types I, II and III). Experiments with individual proteases showed that their activities were strongly inhibited (40–85%) by 5 mM EDTA, indicating that they were metalloproteases. The enzymes exhibited different susceptibilities to inhibition by either 3 mM phenylmethylsulphonyl-fluoride (PMSF), 5 mM thimerosal, or 10 mM cysteine, which respectively inhibit serine, thiol and metalloproteases.     

Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

Evidence for controlled autoproteolysis of alkaline protease. A mechanism for physiological regulation of conidial discharge in Conidiobolus coronatus.

The alkaline serine protease of Conidiobolus coronatus was shown to be involved in its conidial discharge [Phadatare, S., Srinivasan, M. C., Deshpande, M. (1989) Arch. Microbiol. 153, 47-49]. To understand the regulation of conidial discharge, the mechanism of control of protease activity was investigated, which revealed the presence of two electrophoretically separable intracellular proteases (protease I and protease II). The formation of smaller and less-active protease II coincided with the decrease in conidial discharge. In order to trace the origin of protease II, the corresponding purified extracellular enzymes were compared with respect to their biochemical, physiochemical and immunological properties. The biochemical properties, such as optimum pH and temperature, stability, sensitivity to metal ions and substrate specificity were closely similar for both proteases. Amino acid analysis revealed that protease II is completely similar to protease I, though protease I contains an additional portion which is not contained in protease II. Western-blot ELISA, immunotitration and determination of antigenic valencies also revealed the structural similarity between the two proteases. Purified protease I showed partial degradation to protease II in vitro, the process being sensitive to phenylmethylsulfonyl fluoride, indicating its proteolytic nature. These results suggest that the formation of a less-active protease by autoproteolysis represents a novel means of physiological regulation of protease activity, which in turn regulates the conidial discharge in C. coronatus.     

Comparative characterization of two serine endopeptidases from Nocardiopsis sp. NCIM 5124

A protease-producing, crude oil degrading marine isolate was identified as Nocardiopsis sp. on the basis of the morphology, cell wall composition, mycolic acid analysis and DNA base composition. The Nocardiopsis produces two extracellular proteases, both of which are alkaline serine endopeptidases. Protease I was purified to homogeneity by chromatography on CM-Sephadex at pH 5.0 and pH 9.0. Protease II was purified using DEAE-cellulose, Sephadex G-50, phenyl-Sepharose and hydroxyapatite chromatography. Protease I and II had almost similar M(r) of 21 kDa (Protease I) and 23 kDa (Protease II), pI of 8.3 and 7.0 respectively with pH and temperature optima for activity between 10.0 and 11.0 and about 60 degrees C. Specific activities were 152 and 14 U/mg respectively on casein. However, Protease I was antigenically unrelated to Protease II. Both proteases were endopeptidases and required extended substrate binding for catalysis. Both proteases had collagenolytic and fibrinolytic activity but only Protease I had elastinolytic activity. The proteases were chymotrypsin-like with respect to their amino acid compositions and N-terminal sequences.     

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001     

Proteases of the genus Bacillus. II. Alkaline proteases

The alkaline proteases of B. subtilis NRRL B3411, B. pumilis, and B. licheniformis have been isolated by fractionation followed by ion exchange chromatography and their homogeneity demonstrated. General enzyme properties of the B. sublitis NRRL B3411 alkaline protease have been studied and attempts made to differentiate a group of alkaline proteases. It is clear that the alkaline proteases known as Subtilisins or Subtilopeptidases are not, exclusive to B. subtilis but are common to many Bacilli and therefore the generic name Bacillopeptidases has been proposed. It is clear too that on the basis of the effect of pH on activity, amino acid composition, esterase activity, and immunological cross-reactions the Bacillopeptidases can be divided into two groups or types: (a) Bacillopcptidase A (Subtilisin A or Subtilopeptidase A) which includes Subtilisin Carlsberg, B. licheniformis, and B. pumilis alkaline proteases; ( b ) Bacillopeptidase B (Subtilisin B or Subtilopeptidase B) which includes B subtilis NRRL B3411, Subtilisin Novo, Subtilisin BPN' (Nagarse), alkaline protease Daiwa Kasei, and (probably) B. subtilis var. amylosacchariticus. At present, no further differentiation is possible and whether or not the enzymes within group A or B are identical remains an open question. Methods for examination of crude enzyme mixtures or fermentation beers are described and from the examination of a number of crude enzymes and fermentation beers it appears that organisms producing Bacillopeptidase A do not produce neutral protease or amylase, while organisms producing Bacillopeptidase B produce a neutral protease and amylase as well.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg²⁺ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.     

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Extracts from white croaker skeletal muscle showed two alkaline proteases and a trypsin inhibitor when they were chromatographed in DEAE-Sephacel. The activity against azocasein was maximal at pH 8.5 and 9.1 for proteases I and II, respectively. Both enzymes showed optimum activity at 60° C. The molecular masses were found to be 132 kDa for protease 1,363 kDa for protease II, and 65 kDa for the inhibitor. Protease I showed the characteristics of a trypsin-like enzyme, and protease II those of a SH-enzyme. These proteins may play important roles in mechanisms of cellular proteolysis.     

The purification and properties of a fibrinolytic neutral metalloendopeptidase from Streptococcus faecalis

A protease has been isolated by affinity chromatography from culture filtrates of a strain of Streptococcus faecalis previously shown to produce a flbrinolytic enzyme. The pH optimum, molecular weight, metal ion chelator sensitivity, and peptidase specificity place this enzyme in the class of bacterial neutral metalloendopeptidase typified by thermolysin and the Bacillus subtilis neutral proteases. Differences with respect to chemical modification and thermal stability exist between the S. faecalis enzyme and the latter proteases. The S. faecalis enzyme (designated EM 19000) renders fibrinogen incoagulable by degradation of the B (β) chains.