Philibertain g I, the Most Basic Cysteine Endopeptidase Purified from the Latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae)

A new papain-like cysteine peptidase isolated from latex of Philibertia gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7–8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. K_m was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica papaya endopepetidases.     

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.     

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.     

Hieronymain I,a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae)

A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5–9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.     

Morrenain b I,a papain-like endopeptidase from the latex of Morrenia brachystephana Griseb. (Asclepiadaceae)

A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme.     

Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria

Ervatamia coronaria, a flowering plant (family Apocynaceae) indigenous to India, has medicinally important applications. A search for biochemical constituents of the latex of the plant yielded at least three thiol proteases with distinctly different properties. One of them, a highly active protease (ervatamin A), was purified to homogeneity by ion exchange and gel filtration chromatography. The enzyme exhibited high proteolytic activity toward natural substrates and amidolytic activity toward synthetic substrates. The pH and temperature optima for proteolytic activity were 8–8.5 and 50–55°C, respectively. Proteolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. The estimated molecular mass of the enzyme was 27.6 kDa. The extinction coefficient (1% 280) of the enzyme was estimated as 21.9, and the protein molecule consists of 8 tryptophan, 11 tyrosine and 7 cysteine residues. Isoelectric point of the purified enzyme was 8.37. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and a typical color in ELISA. The N-terminal sequence of the enzyme showed conserved amino acid residues to other plant cysteine proteases. Ervatamin A shows high activity in relation to the other thiol proteases isolated from the same source.     

Procerain,a stable cysteine protease from the latex of Calotropis procera

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 g of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (M_r = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.     

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.     

Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

Indicain, a dimeric serine protease from Morus indica cv. K2

A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.     

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.     

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.     

Isolation,Purification, and Properties of Cysteine Proteinase from <Emphasis Type="Italic">Bombyx mori</Emphasis> L. Eggs

Silkworm moth (bombyx) egg cysteine proteinase with maximal activity at pH 3.0 was purified by chromatography and isoelectrofocusing. On SDS-electrophoresis in polyacrylamide gel the purified enzyme showed a single band of molecular mass 50 kD. Isoelectrofocusing revealed that the bombyx egg cysteine proteinase exists in two forms with pI values of 5.95 and 6.43, respectively. The enzyme activity was sensitive to inhibition by iodoacetamide and p-chloromercuribenzoate but resistant to EDTA, pepstatin, and phenylmethylsulfonyl fluoride. The cysteine proteinase hydrolyzes storage proteins of bombyx eggs but it was inactive with respect to N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA). It is a cathepsin L-like enzyme.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.     

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from <Emphasis Type="Italic">Asclepias fruticosa</Emphasis> latex

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.