Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera.     

Isolation of Alkaline Protease Inhibitor Producing Microorganisms

For the purpose of obtaining microorganisms capable of producing alkaline protease inhibitor, screening test was carried out. One strain of microorganisms (No. S–3253) showed strong ability to produce alkaline protease inhibitor.

The morphological and physiological characteristics of strain S–3253 were studied. This strain was found to belong to Streptomyces albogriseolus.

When the strain S–3253 was cultivated at 45°C with a medium containing 4% polypepton, 2% starch, 0.1% yeast extract, 0.1% NaCl, 0.1% K₂HPO₄ and 0.05% MgSO₄-7H₂O in shake-flasks (pH 7.0), the highest activity was obtained after 40~48 hr cultivation.     

Plasmodium falciparum: Differential Sensitivity In Vitro to E-64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)

ABSTRACT We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 μM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 μM) or 12 h at the inhibitory concentration 50% (12 μM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 μM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

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.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

Effect of a Microbial Acid Protease Inhibitor (S-PI) on the Production of Fungal Acid Protease

Cladosporium sp. No. 45–2, an acid protease-producing microorganism, was cultured in medium containing a microbial acid protease inhibitor (S–PI). By the addition of S–PI, the amount of acid protease in the culture broth showed an increase of 50~80% over those of normal culture (S–PI-free). Acid protease was purified from the S–PI-added culture filtrate, and its enzymatic and physicochemical properties were compared with those of acid protease obtained from normal culture. It was determined that the acid protease obtained from S–PI-added culture was the same as that of normal culture, but that the productivity was increased by the addition of S–PI.

The increase in acid protease productivity is assumed to be due to a change in metabolism by the addition of S–PI.     

Protease from bovine spleen with kininogenase activity]

A protease with kininogenase activity at pH 7.5 was isolated from bovine spleen extract by gel filtration and ion exchange chromatography. The protease was found in the fraction with molecular weight lower than 25.000 and was separated from the other neutral SH-dependent protease by chromatography on KM-cellulose. The kininogenase activity was inhibited by DFP and trasylol; soybean trypsin inhibitor had no effect. The protease did not split N-benzoyl-L-arginine ethyl ester and N-benzoyl-D, L-arginine p-nitroanilide.     

Purification and Properties of Mucor pusillus Acid Protease

The protease produced by Mucor pusillus was recovered from a wheat bran medium by treatment with ammonium sulfate, ethyl alcohol, gel filtration and ion-exchange chromatography. The yield of the enzyme was 55%. The overall increase in the specific activity of the protease was 34-fold. The purified protease was most active at pH 3.8 and 5.6 against hemoglobin and casein, respectively. Optimal hydrolysis of casein was observed at 55 C. The enzyme was stable from pH 3.0 to 6.0. Enzyme inactivated by metal ions was reactivated by ethylenediaminetetraacetate and o-phenanthroline. Reducing agents and thiol poisons had no effect on the protease, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inhibit the protease, indicating the probable absence of serine in the active center. The Michaelis-Menten constant for casein was 0.357%. Electrophoretic analysis of active protein recovered by ion-exchange chromatography showed that the protease preparation was homogeneous.     

Studies on Bacterial Protease

A neutral protease of Bacillus subtilis var. amylosacchariticus was purified and crystallized by sequential chromatography on columns of Duolite A-2 anion-exchange resin, CM-cellulose and DEAE-sephadex A-50. The crystalline preparation was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria. The enzyme was most active at near pH 7 against casein and stabilized by calcium salts. Some metalchelating agents and metal ions such as Hg?, Pb?, Cu? and Fe? markedly inactivated the enzyme, whereas diisopropyl phosphorofluoridate, sulfhydryl reagents and protease inhibitor of potato did not affect the activity. The neutral protease obtained here was rather stable as compared with the neutral protease ever reported and was able to be freeze-dried without any appreciable lose in enzyme activity.     

Purification and Characterization of Microbial Protease Produced Extracellularly from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺ and K⁺, but 10 mM Fe³⁺ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).     

Protease inhibitors and proteolytic signalling cascades in insects

Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.     

Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Distribution of Acid Protease Activity in the Squid Nervous System

Abstract: Acid protease activity was measured in homogenized stellate ganglion, axoplasm extruded from the squid giant axon, homogenized fin nerves, and in lysed synaptosomes prepared from the optic lobe of the squid. At least two different acid protease classes were distinguished on the bases of their inhibitor profiles. Acid protease activity was present in each of the above tissues except extruded axoplasm. This result suggests that the acid protease activity found in our homogenized finnerves might be located not within the axons but rather in glial cells or extracellular tissue. The absence of acid protease activity in extruded axoplasm indicates that acid proteases are unlikely to play a significant role in the catabolism of intracellular proteins along the length of the axon.     

交联琼脂糖珠固定化蛋白酶在纯化牛肺Kunitz抑制剂中的应用

本文介绍了珠状交联琼脂糖及以此作为载体,经氯代环氧丙烷活化后与蛋白酶(胰蛋白酶或糜蛋白酶)结合,制成固定化蛋白酶亲和吸附剂,进而用以亲和层析牛肺提取液中的Kunitz抑制剂的方法。纯化出的抑制剂在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带,与参照物Trasytol(商品Kunitz抑制剂)具有相对应的电泳迁移率,其分子量也相符。纯化产品每毫克蛋白的抑制活力相当于16 000胰蛋白酶BAEE单位。纯化效果为90倍,收率约85％。     

一种苦荞麦种子蛋白酶抑制剂的纯化、特性及其抗虫活性

蛋白酶抑制剂广泛存在于生物体内, 是自然界含量最为丰富且具有一定防御作用的蛋白种类之一. 本文采用离子交换层析和凝胶层析等方法,从苦荞麦种子中分离出一种胰蛋白酶抑制剂（TBTI-Ⅱ）. SDS-PAGE分析表明,TBTI Ⅱ的分子量约9.0 kD,由80个氨基酸残基组成,分子中含有较多的 Glu, Asp 和Arg. TBTI-Ⅱ具有较高热稳定性.当在100℃加热处理10 min后,仍保留有67.6%的抑制剂活性. 动力学测定显示,来自苦荞麦中的TBTI-Ⅱ对胰蛋白酶的抑制作用常数(Ki)为1.01×10－4 mol/L. 另外,将含有不同活力单位的苦荞麦蛋白酶抑制剂掺入到棉铃虫的饲料中进行饲养试验显示,TBTI-Ⅱ具有明显的抑制棉铃虫生长的作用. 这些结果表明,来自苦荞麦种子中的小分子蛋白酶抑制剂可能是一种潜在的抗虫因子.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.     

Detection of an Intracellular Protease Inhibitor in Archaea

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a ∼90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea. Received: 4 May 2002 / Accepted: 10 July 2002     

A Protease Inhibitor from Penicillium cyclopium

When the protease inhibitor from Penicillium cyclopium was mixed with the acid protease of the mold at acid pH formed a precipitate consisting of a enzyme-inhibitor complex. The precipitation occurred maximally at pH 3.0 and was interfered with by increasing amounts of salts and other protein. Subsequent incubation of the complex brought about inactivation of the enzyme and the inactivation was found to be accompanied by modification of the enzyme so that less was precipitable with trichloroacetic acid. Paper chromatography revealed that the enzyme on complete inactivation had been degraded to several fragments or polypeptides. The inhibitor acted on the enzyme in a catalytic fashion, bringing about degradation of more than a stoichiometric amount of enzyme. The proposed mechanism of the inhibitor action involved acceleration of auto-digestion of the enzyme which splits the molecule into small fragments and abolishes the activity.