BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.     

A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.     

Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum

Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C.     

Purification and properties of arginine esterases from Bitis arietans (puff adder) venom

An arginine esterase (FT1) was purified from B. arietans venom by gel-filtration and ion-exchange chromatography. The purified enzyme contains 21.6% of carbohydrate, 240 amino acids including 12 half-cystine residues and has a mol. wt of approximately 43,000. The purified enzyme has a high esterolytic activity towards N-alpha-benzoyl-L-arginine ethyl ester but shows no proteolytic activity against Azocoll and no clotting activity with fibrinogen. The N-terminal sequence of the arginine esterase from B. arietans venom shares a significant degree of sequence homology with the arginine esterase of B. nasicornis, the thrombin-like enzyme of C. adamanteus and the kallikrein-like enzymes of C. atrox venoms. It would appear that the arginine esterase from B. arietans venom exists in various multiple forms of the enzyme.     

Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.     

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.     

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG‐75, DEAE‐Sephadex A‐50, and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). VLCII as a thrombin‐like enzyme was able to hydrolyze Nα‐CBZ L‐arginine‐p‐nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10‐phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration‐dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.     

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.     

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. CONCLUSIONS: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.     

木瓜蛋白酶PPAⅢ自水解作用

木瓜蛋白酶ＰＰＡⅢ自水解作用王秀艳,周慧,葛玉斌,李惟（吉林大学分子生物学系,长春１３００２３）ＰＰＡⅢ、ｐａｐａｉｎ、ｐｅｐｓｉｎｅ、ｓｔｅｍｂｒｏｍｅｌａｉｎ等都属于以Ｃｙｓ－ＳＨ为活性中心的酶,同种分子之间存在着一种相互切割的趋势,称为酶自水解...     

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.     

Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomus papatasi (Diptera, Psychodidae)

Phlebotomus papatasi is susceptible to Leishmania major which it transmits in nature, but is resistant to L. donovani. The present study compares the effect of L. major and L. donovani on the proteolytic activity of P. papatasi gut enzymes. The experiments measured digestion of C14-labeled globin by gut homogenates of flies. Homogenates were prepared from flies fed on serum only (controls) or from flies fed serum containing promastigotes or their dried culture overlayer. In other experiments, the promastigotes or dried culture overlayers were added in vitro to the gut homogenate of control flies. Proteolytic activity of gut homogenate from flies infected with L. major was about one-third less than that of controls, while that from flies infected with L. donovani was one-third greater. Ingestion of L. major dried culture overlayer had an effect on flies similar to that of the promastigotes, while L. donovani dried culture overlayer produced no significant effect. When added to gut homogenate in vitro, promastigotes of both species promoted proteolysis as did dried culture overlayer of L. major. Dried culture overlayer of L. donovani, however, had an opposite effect. It is suggested that the observed reduction in proteolytic activity caused by L. major infection may result from inhibition of enzyme production.     

Lymphocyte suppressive peptides from fibrinogen are derived predominantly from the A alpha chain

Cellular immune responses can elicit local deposition of fibrin at the site of immunologic reactions, as well as the formation of intravascular fibrin in disseminated reactions. The subsequent physiologic proteolysis of fibrinogen and fibrin by plasmin results in small peptides that suppress lymphocyte functions in vitro and in the immune response in vivo. The intramolecular origin of lymphocyte suppressive activity and the proteolytic events responsible for the release of active peptides have been analyzed. Plasmic peptides from the isolated B beta and gamma constituent chains of fibrinogen did not inhibit mitogen-driven responses of human peripheral blood mononuclear cells. In contrast, plasmic digests of the A alpha chain, but not the intact A alpha chain were suppressive. Advanced plasmic digests of fibrinogen and the A alpha chain were suppressive at similar concentrations, suggesting that biological activity is derived predominantly from the A alpha chain. Limited plasmic digests of fibrinogen were fractionated to yield a heat-precipitable 250,000 dalton fragment X and heat-soluble proteolytic products containing fragments derived from the carboxyl-terminal region of the A alpha chain including a 42,000 dalton major A alpha chain derivative. Neither fragment X nor derivatives produced by its additional plasmic proteolysis were suppressive. In contrast, the heat-soluble fraction from limited plasmic cleavage was suppressive, and this activity was enhanced 10-fold by additional plasmic cleavage of this fraction. The isolated 42,000 dalton A alpha chain fragment was devoid of activity, but plasmic digestion of this derivative generated peptides of less than 8000 daltons that inhibited mitogen-stimulated thymidine uptake by lymphocytes. Two synthetic peptides corresponding to A alpha 220-230 and B beta 43-47, peptides with known vasoactive activities, suppressed lymphocyte thymidine uptake at very high concentrations. Based on their maximal yield from plasmic digests of fibrinogen, these two peptides would account for only 1% of the immunosuppressive activity of fibrinogen derivatives. In summary, the results indicate that the suppressive activity of fibrinogen is predominantly derived from the 42,000 dalton carboxyl terminal region of the A alpha chain of the molecule and is not attributable to the known vasoactive peptides. Initial proteolytic release of this region from the core of fibrinogen does not result in suppressive activity, but additional cleavage releases small peptides with the lymphocyte inhibitory function.     

The action of gamma irradiation on terrilytin immobilized on cellulose-fiber materials

The effect of gamma-radiation on terrilytin, a proteolytic enzyme immobilized on modified and nonmodified cellulose materials was studied by EPR. Dialdehyde cellulose and graft copolymer of cellulose and polyacrylic acid were used as the modified cellulose materials. Dependence of the native and immobilized terrilytin activity and the content of free radicals in the irradiated samples on the irradiation dose was observed. It was shown that immobilization of the enzyme led to increasing of its stability to the effect of the ionizing radiation. This was due to transfer of the free valency from terrilytin to the carrying polymer which prevented radiation and chemical destruction of the enzyme. The proteolytic activity of native terrilytin subjected to gamma-irradiation markedly decreased because of intramolecular and intermolecular interactions during reactions of the terrilytin free radicals, since in this case there was no polymer as an acceptor of the enzyme free valency.     

Immobilized α-chymotrypsin: Pore diffusion control owing to pH gradients in the catalyst particles

The fast enzymatic hydrolysis of D ,L -phenylalanine methylester (DLE) to L -phenylalanine (LA) and D -phenylalanine methylester (DE) with immobilized α-chymotrypsin was chosen as a model reaction. Under the experimental conditions applied in the present investigations the pore diffusion is the rate-limiting step of this reaction owing to the pH gradient in the particles. The effectiveness of the catalyst is experimentally determined as a function of the substrate concentration based on measurements of the enzyme protein content of native and immobilized enzyme. The proteolytic reaction is theoretically treated by also using a pore diffusion model which takes into account the concentration gradients of substrate and product, pH- and enzyme activity profiles, as well as the change of buffer capacity of the solute in the catalyst particles. The model parameters were experimentally determined for the investigated system. It can be shown that conditions are possible for which the effectiveness of the catalyst exceeds unity.     

Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox.

Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.     

Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.     

Proteolysis coupled with ATP. Regulation of activity of proteolytic centers of Escherichia coli lon protease

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP-Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by the native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of the intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

Purification and partial characterization of a new proteolytic enzyme from the venom of Bothrops moojeni (CAISSACA).

A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.