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.     

Studies on the Protease Constitution of Aspergillus oryzae

In order to elucidate the protease constitution of Aspergillus oryzae, systematic separation of proteases was elaborated by sequential chromatography on Amberlite CG–50, DEAE-Sephadex A–50 and CM-cellulose. As the results, three kinds of proteases, that is, acid-, neutral- and alkaline proteases were isolated and purified in crystalline form except neutral one. Purified neutral protease could not be crystallized, but was confirmed to be homogeneous by ultracentrifugal analysis. Besides these proteases, a new protease which was unknown up to the present in the constitution of Asp. oryzae proteases, was first isolated and designated as “semi-alkaline protease” according to its optimal pH.     

Properties of extremely thermostable proteases from anaerobic hyperthermophilic bacteria

Summary Hyperthermostable proteases were characterized from five archaeobacterial species (Thermococcus celer, T. stetteri, Thermococcus strain AN 1, T. litoralis, Staphylothermus marinus) and the hyperthermophilic eubacterium Thermobacteroides proteolyticus. These proteases, which were found to be of the serine type, exhibited a preference for phenylalanine in the carboxylic side of the peptide. The enzymes from Thermococcus stetteri and T. litoralis hydrolysed most substrates (peptides) tested. All proteases were extremely thermostable and demonstrated optimal activities between 80 and 95°C. The pH optimum was either neutral (T. celer, Thermococcus strain AN 1) or alkaline. The protease of Thermobacteroides proteolyticus was optimally active at pH 9.5. Zymogram staining showed the presence of multiple protease bands for all strains investigated.Offprint requests to: G. Antranikian     

Preparation and Purification of Microbial Proteases With Special Reference to The Bacilli

Methods for the examination of bacteria for protease production on semisolid media are described. The selection of media for production of small quantities of crude bacterial proteases from pure cultures of selected microorganisms in shake flasks is discussed. The most useful media have been found to be a grain-based medium, a soya fluff-starch-yeast extract medium and a fish meal-enzose-cerelose-cornsteep liquor medium. The alkaline proteases and neutral proteases can be identified and differentiated by specific assays and a purification procedure planned dependent upon the enzymes present in the fermentation beer. Crude enzyme can be precipitated from the fermentation beer by the addition of organic solvents such as acetone or isopropanol or by the addition of salts such as ammonium or sodium sulfate. The alkaline proteases typified by B. subtilis alkaline protease can be extensively purified by chromatography on Duolite C-10 cation exchange resin, whereas the neutral protease of 3 subtilis is best purified by chromatography on hydroxylapatite. Methods for purification of other proteases are discussed and the prechromatography steps for removal of pigment and other gross impurities are described.     

A new thermostable proteinase from Thermoactinomyces SP.27a. Cloning and gene expression

A library of Thermoactinomyces sp. 27a, producer of thermostable proteases of different groups, has been created. Gene coding for thermostable neutral proteinase was cloned and expressed in Bac. subtilis cells. Restriction map for cloned DNA fragment was created and physicochemical parameters of recombinant proteinase were characterized. The thermostability and optimum of proteolytic activity of the enzyme was lower than in the natural Thermoactinomyces sp. strain, which can be due to heterologous expression of the gene coding for thermostable protein in the mesophilic host.     

Characteristics of a highly thermostable neutral protease produced fromBacillus stearothermophilus

A highly thermostable neutral protease was found in culture filtrates ofBacillus stearothermophilus. The optimum reaction pH and temperature of this protease were 6.0 and 60°C, respectively, and 90% activity remained even after heat treatment at 90°C for 30 min. The protease was markedly inactivated by diisopropyl fluorophosphate, but EDTA and iodoacetic acid hardly affected it. The neutral protease therefore could be defined as a highly thermostable, neutral(-serine) protease.     

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.     

Purification and properties of proteases produced byalternaria alternata (Fr.) Keissl,regulation of production by fructose

A strain ofAlternaria alternata (Fr.) Keissl, when grown on wheat bran Czapek Dox medium was found to secrete one neutral and two alkaline proteases. The purified enzymes were found to be endo peptidases, the alkaline proteases being serine proteases and neutral proteases being cysteine proteases. Fructose when added to the culture medium was found to give rise to a new neutral protease at the expense of the neutral protease produced in the absence of fructose and was also found to enhance the production of alkaline proteases. It also appears that fructose modifies the alkaline proteases with respect to some characteristics such asV _max, Ea etc. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis indicated a significantly altered protein profile in fructose supplemented medium.     

Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33

Three thermostable proteases, designated S, N, and B, are extracellular enzymes produced by Bacillus stearothermophilus strain TLS33. They were purified by lysine affinity chromatography, strong anion exchange Q HyperD chromatography, and Ultrogel AcA44 gel filtration. The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 36, 53, and 71 kDa, respectively. Thermostable protease S bound strongly to the lysine affinity column and could be purified by this single step. The optimum pH values of proteases S, N, and B were shown to be 8.5, 7.5, and 7.0, respectively. The maximum activities for the enzymes were at 70, 85, and 90 degrees C, respectively. Proteases S, N, and B at pH 7.0 in the presence of 5 mM CaCl(2) retained half their activities after 30 min at 72, 78, and 90 degrees C, respectively. All three thermostable proteases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline, and the proteolytic activities were restored by addition of ZnCl(2). They can thus be classified as Zn(2+) metalloproteases. The cleavage specificities of proteases S, N, and B on a 30-residue synthetic peptide from pro-BPN' subtilisin were Tyr-Ile, Phe-Lys, and Gly-Phe, respectively.     

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.     

Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

Background  

Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia.     

Complete Amino Acid Sequence of Neutral Protease from Bacillus subtilis var. amylosacchariticus

The neutral protease of Bacillus subtilis var. amylosacchariticus was cleaved chemically or digested with proteolytic enzymes, and the resultant peptides were separated and purified by high performance liquid chromatography. The sequence analyses of these peptides by the manual Edman procedure established the complete amino acid sequence of the enzyme. The neutral protease consisted of 300 amino acid residues with Ala and Leu as its amino- and carboxyl-termini, respectively, and the molecular weight was calculated to be 32,633. The sequence was found to be identical to that of B. subtilis 1A72 neutral protease, which was deduced from nucleotide sequencing. Comparison of the sequence with those of other Bacillus proteases revealed that the putative active site amino acid residues, Zn-binding ligands, and two Ca-binding sites were well conserved among them, as compared with those of thermolysin.     

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.     

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.     

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.     

A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases

A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350-361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.     

Cloning of the gene responsible for the extracellular proteolytic activities of Bacillus licheniformis

Summary Effect of the cloned gene of Bacillus licheniformis on the extracellular proteolytic activities of B. subtilis was investigated. The gene was cloned onto the vector plasmid pUB110 (3.0 Md), and the introduction of the hybrid plasmid [pAN2 (5.4 Md)] into the cells of B. subtilis resulted in a marked increase of activities of the extracellular alkaline and neutral proteases, which had optimal pHs at 10.5 and 7.2, respectively. On DEAE-Sephadex column chromatography, the extracellular activity of B. subtilis with pAN2 was separated into two active fractions (a₁ and b₁). The activity in a₁ was specifically inactivated by diisopropyl phosphorofluoridate (DFP) and tosyl fluoride (TSF), potent inhibitors of alkaline proteases, while, the activitiy in b₁ was inhibited by ethylenediaminetetraacetate (EDTA), an inhibitor of neutral protease, but not by DEP or TSF.Sub-cloning with genes shortened to about 0.85 Md (pAN2-1) and 0.25 Md (pAN2-2) increased the activities of both alkaline and neutral proteases. The extracellular -amylase and ribonuclease production was also increased when the host strain was transformed with these hybrid plasmids (pAN2, pAN2-1, pAN2-2). The increase in activity of proteases by the cloning was discussed in relation to regulation of the production and/or secretion of the enzyme.     

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.     

Production of neutral and alkaline proteases in batch and chemostat cultures by bacillus mesentericus 76

The kinetics of the bacterial extracellular protease synthesis (neutral and alkaline protease of Bacillus mesentericusstrain 76, R-form) in batch and chemostat cultures under conditions of glucose limitation were investigated. When the medium was supplemented with casein the production of the proteases was significantly higher. Optimal dilution rates for obtaining of two proteases are fixed. The synthesis of both alkaline and neutral proteases is controlled by catabolite repression and induction.