Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Isolation and Characterization of a Cysteine Protease of Freesia Corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M _r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

Studies on the Lipase of Thermophilic Fungus Humicola lanuginosa

A protease occurring in the endosperm fraction of germinating corn was purified by means of (NH₄)₂SO₄ fractionation, CM-celluIose chromatography, DEAE-cellulose chromatography, Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified protease was found to have a molecular weight of about 21,000 and an isoelectric point of pH 2.3 or lower. The optimum pH was found to lie at 3.0 when measured with denatured hemoglobin as substrate. The protease was generally activated by thiol compounds and completely inhibited by p-chloromercuribenzoic acid. Neither diisopropylphosphofluoridate nor diazoacetyl-dl-norleucine methyl ester affected the protease activity. Antipain greatly inhibited the protease action whereas pepstatin had no significant effect. These data indicate, in conclusion, that the protease possesses a unique property to be a sulfhydryl enzyme most active in an acidic region around pH 3.     

Alkaline Serine Protease Is an Exotoxin of Vibrio alginolyticus in Kuruma Prawn,Penaeus japonicus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose High Performance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF), and showed maximal activity at pH 10, having a molecular weight of about 33 kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl₂ but partially inhibited by CaCl₂, CuCl₂, CoCl₂, MnCl₂, and ZnCl₂, and not inhibited by ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD₅₀ values of 0.30 and 0.27 μg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy. Received: 12 April 1996 / Accepted: 31 July 1996     

Purification and Characterization of Vibrio vulnificus Protease

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30%. The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.     

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.     

Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus

Summary The proteolytic activity produced by a new species of Bacillus isolated in our laboratory was investigated. This enzyme was purified to homogeneity from cell-free culture liquids of B. thermoruber. The purification procedure included ion-exchange chromatography on DEAE-Sephadex A-50 and -casein agarose affinity chromatography. The protease consists of one polypeptide chain with a molecular weight of 39000±800. the isoelectric point was 5.3; the optimum pH and temperature for proteolytic activity (on casein) was found to be pH 9 and 45°C respectively. Enzyme activity was inhibited by PMSF and EDTA. The stability was considerably increased by addition of Ca²⁺, and the protease exhibited a relatively high thermal stability. The alkaline protease shows a preference for leucine in the carboxylic side of the peptide bond of the substrate. The K _m value for benzyloxycarbonyl-Ala-Ala-Leu-p-nitroanilide was 2.5 mM.     

Novel cathepsin L-like protease from dermestid beetle Dermestes frischii maggot

A novel cathepsin L-like protease from dermestid beetle Dermestes frischii maggot guts was obtained and investigated. The protease was isolated through affinity chromatography at arginine-diasorb followed by FPLC gel-filtration at Superdex 75. Protease is active against chromogenic peptide substrates, containing Arg or Leu in P1 position and a hydrophobic residue in P2 position. PH optimum is about 4,5 and temperature optimum at 40 °C. Enzyme is inhibited completely by HgCl₂ and leupeptin that prove it’s belonging to cysteine proteases of papain family.cDNA analysis of cathepsin L-like protease showed that protein sequence consists of 339 amino acid residues. Mature cysteine protease contains 219 amino acid residues corresponding to molecular mass 24027.20 Da. Residues of the active site were identified: Gln¹⁴⁰, Cys¹⁴⁶, His²⁸⁵, Asn³⁰⁶ and Trp³⁰⁸. Calculated pI is 4,73. The amino acid sequence of the cystein protease from dermestid beetle displays high structural homology with cathepsin L of other insects.     

Purification and some properties of an extracellular acid protease from <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">clavatus</Emphasis>

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH₄)₂SO₄ fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg⁺² and partially inhibited by Cu⁺², Zn⁺² and Mn⁺². The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a K_i of 7.8 μM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.     

Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH₂-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofM _r 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.     

Comparison of Phytolacain G,a Cysteine Protease from Fruit of Phytolacca americana,with Phytolacain R

The enzymatic properties of phytolacain G, a protease isolated from green fruit of pokeweed, were compared with those of phytolacain R, a protease obtained from ripe fruit. The optimum pH of phytolacain G was 7.5-8.0 at 37°C using casein as the substrate. The enzyme was strongly inhibited by iodoacetic acid and p-chloromercuribenzoic acid, but not by diisopropyl fluorophosphate or EDTA. These results indicated that phytolacain G was a cysteine protease, like phytolacain R. Nine sites of oxidized insulin B-chain were cleaved by phytolacain G during 20 h of hydrolysis. The six sites cleaved by phytolacain G were also cleaved by phytolacain R. The substrate specificity of phytolacain G was broad, but the preference for hydrophobic residues at the P₂ position was similar to the substrate specificity of papain. The amino-terminal sequence of phytolacain G was not identical with that of phytolacain R; however, the amino acid residues conserved in the papain family were also conserved in this enzyme.     

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum)

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl₂ buffer (pH 8.2) with a flow rate of 0.5?mL min^?1. The molecular weight and purity of ～23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg^?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.     

Characterization of a novel cysteine peptidase from tissue culture of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary A simple and efficient medium for callus tissue culture from garlic to obtain maximal proteolytic activity is described. Murashige and Skoog basal medium supplemented with 4.44 μM naphthaleneacetic acid (NAA) and 0.54 μM benzyladenine (BA) resulted in the best biomass production and protease expression. The protease activity belongs to the class of cysteine proteases since they are inhibited by E₆₄ and Leupeptin and also they are activated by 2-mercaptoethanol and cysteine. They showed good thermal stability. Three active protease bands were found in zymograms of Allium sativum. The in vitro system revealed a significantly higher protease level than storage and embryo tissues of in vivo bulbs.     

Studies on Protein Metabolism in Higher Plants

A leaf protease of tobacco whose activity was enhanced during curing was purified about 60 times with ammonium sulfate fractionation, ethanol precipitation, calcium phosphate gel treatment and Sephadex G-200 column chromatography, and some properties of the protease were examined. The purified enzyme showed the optimum pH at 5.5 and the optimum temperature at 60°C. The protease activity was stable between pH 4.5 and 5.5 at 50°G or at pH 5.5 below 40°C for 1 hr, but completely destroyed at 70°C during 1 hr. The protease activity was greatly activated by reducing agents such as cysteine, glutathione or mercaptoethanol and inhibited by p-chloromercuribenzoate, phenyl- mercuric acetate or silver ions. Metal ions except for silver ion and ethylenediamine tetraacetic acid did not affect the protease activity so far examined.     

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits

A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl₂, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 M. When assayed on N--CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.     

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated<Emphasis Type="Italic">Bacillus subtilis</Emphasis> PE-11

The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca²⁺, Mg⁺², and Mn⁺². Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl₂ and 1M glycine. The addition of 10mM CaCl₂ and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl₂ and 1M glycine.     

Purification and Characterization of Metallo-β-Lactamase from Serratia marcescens

Carbapenem-hydrolyzing β-lactamase from Serratia marcescens FHSM4055 was purified 926-fold by means of carboxylmethyl Sephadex C-50, Sephacryl S-200, and Mono S column chromatography. The molecular weight was 30,000 by SDS-PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p-chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo-β-lactamase and that the SH-group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (k_cat/K_m) showed that the enzyme hydrolyzed various β-lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino- and imino-groups, ceftazidime with a carboxypropyloxyimino-group, and cefclidin with a carbamoylquinuclidine-group were poor substrates among the β-lactam antibiotics other than the monobactams tested. The plots of the turnover number (k_cat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK₁, 5.9; pK₂, 9.0; pK₃, 10.8), whereas those of k_cat/K_m gave a bell-shaped curve (pK₁, 5.8; pK₂, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N-terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71-78), the above enzymatic characteristics seem to coincide.     

Extraction,purification and characterization of a novel cysteine protease from the latex of plant <Emphasis Type="Italic">Vallaris solanacea</Emphasis>

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.     

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N--CBZ-amino acid-p-nitrophenyl esters, and the highest k _cat/K _m values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.