Proteinaceous Protease Inhibitors Research Articles

Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

Development of a novel peptide inhibitor of subtilisin BPN'.

Proteinaceous protease inhibitors can strongly and specifically inhibit cognate proteases, but their use as pharmaceuticals is limited by their size. As such, the development of effective protease peptide inhibitors would be beneficial for biochemical studies and drug discovery. In this study, we applied a phage display system to select subtilisin BPN'-binding peptides and evaluated their inhibitory activities against subtilisin BPN'. A 12mer peptide with an intramolecular disulfide bond inhibited subtilisin BPN' (Ki value of 13.0 nm). Further mutational analyses of the peptide resulted in the development of a short peptide inhibitor against subtilisin BPN' that showed high inhibitory activity and binding affinity (Ki value of 0.30 nm). This activity was found to be derived from the conformational rigidity caused by the intramolecular disulfide bond and the small residue at the P1' site and from the interaction of the P4 and P6' residues with subtilisin BPN'.

NMR assignments and secondary structure distribution of emfourin, a novel proteinaceous protease inhibitor.

Emfourin (M4in) from Serratia proteamaculans is a new proteinaceous inhibitor of protealysin-like proteases (PLPs), a subgroup of the well-known and widely represented metallopeptidase M4 family. Although the biological role of PLPs is debatable, data published indicate their involvement in pathogenesis, including bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls. Gene colocalization into a bicistronic operon observed for some PLPs and their inhibitors (as in the case of M4in) implies a mutually consistent functioning of both entities. The originality of the amino acid sequence of M4in suggests it belongs to a previously unknown protein family and this encourages structural studies. In this work, we report a near-complete assignment of 1H, 13C, and 15N resonances of recombinant M4in and its structural-dynamic properties derived from the chemical shifts. According the NMR data analysis, the M4in molecule comprises 3-5 helical elements and 4-6 β-strands, at least two of which are apparently antiparallel, ascribing this obviously globular protein to the α + β structural class. Besides, two disordered regions also exist in the central loops between the regular secondary structural elements. The obtained data provide the basis for determining the high-resolution structure as well as functioning mechanism of M4in that can be used for development of new antibacterial therapeutic strategies.

Small peptides inhibit gut trypsin-like proteases and impair Anticarsia gemmatalis (Lepidoptera: Noctuidae) survival and development.

Anticarsia gemmatalis larvae are key defoliating pests of soybean plants. Inorganic insecticides, harmful to the environment and human health, are the main molecules used in the control of this pest. To apply more sustainable management methods, organic molecules with high specificities, such as proteinaceous protease inhibitors, have been sought. Thus, molecular docking studies, kinetics assays, and biological tests were performed to evaluate the inhibitory activity of two peptides (GORE1 and GORE2) rationally designed to inhibit trypsin-like enzymes, which are the main proteases of A. gemmatalis midgut. The molecular docking simulations revealed critical hydrogen bonding patterns of the peptides with key active site residues of trypsin-like proteases of A. gemmatalis and other Lepidopteran insects. The negative values of binding energy indicate that hydrogen bonds potentiate the tight binding of the peptides with trypsin-like proteases, predicting an effective inhibition. The inhibition's rate constants (Ki) were 0.49 and 0.10 mM for GORE1 and GORE2, resulting in effective inhibition of the activity trypsin on the L-BApNA substrate in the in vitro tests, indicating that the peptide GORE2 has higher inhibitory capacity on the A. gemmatalis trypsins. In addition, the two peptides were determined to be reversible competitive inhibitors. The in vivo test demonstrated that the peptides harm the survival and development of A. gemmatalis larvae. These results suggest that these peptides are potential candidates in the management of A. gemmatalis larvae and provide baseline information for the design of new trypsin-like inhibitors based on peptidomimetic tools. © 2020 Society of Chemical Industry.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legumecropproductivities. Protease inhibitors affectprotein degradation and reducethe availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases andthe growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmataliswas delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage whereinA. gemmatalis fedvoraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline informationabout the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Anti-hyperglycemic Properties of a Purified Proteinaceous Protease Inhibitor from Macrotyloma Uniflorum Seeds

Fabaceae are a rich source of protease inhibitors. A proteinaceous protease inhibitor of 25 KDa designated as Macrotyloma uniflorum protease inhibitor (MUPI) was isolated from seeds of Macrotyloma uniflorum and purified to homogeneity by ammonium sulphate precipitation, DEAE Sepharose column and CNBr activated Sepharose 4B Trypsin affinity chromatography. The purity was checked by reverse phase HPLC. Long-term type 2 diabetes can lead to various biological complications, such as hypertension and heart-related diseases. The glucose uptake studies were carried out with the purified MUPI and it reveals its potential to be explored as a potent anti-hyperglycemic agent. MUPI was rendered safer on HepG2 cells after MTT cytotoxicity assay. The results of glucose uptake studies suggest MUPI to be equally efficient to that of the positive control drug Metformin against diabetes mellitus. The significance of plant-based inhibitors for modulating carbohydrate breakdown and control of glycemic index is explored to reduce the risk factors and side effects of the available drugs.

ANTIMICROBIAL ACTIVITY OF A PROTEASE INHIBITOR ISOLATED FROM THE RHIZOME OF CURCUMA AMADA

Objective: Protease inhibitors (PIs) are effective antimicrobial agents, and this study was aimed to study the antibacterial efficacy of a PI isolated from the rhizome of Curcuma amada.Methods: A proteinaceous protease inhibitor was isolated from the rhizome of C. amada and purified by Sephadex G-50 gel permeation chromatography. The purified inhibitor was denoted as Curcuma amada protease inhibitor (CAPI). The antibacterial effect of CAPI against Gram-positive and Gram-negative bacteria, the minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) of CAPI was studied in vitro, and the membrane disruption activity of CAPI was also analyzed.Results: CAPI was effective against both Gram-positive and Gram-negative bacteria, with slightly higher concentrations required for Gram-negative bacteria. The MIC ranged from 75 to 100 μg/ml and the MBC ranged from 100 to 125 μg/ml of CAPI. The study of membrane disruption by CAPI revealed the release of cell contents, namely, reducing sugars and proteins from the bacterial cell.Conclusion: A PI was effectively isolated from the rhizome of C. amada, and the isolated inhibitor proved to be a promising antibacterial agent.

Isolation, purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine Pseudomonas mendocina.

An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina. BTPI-301was purified to homogeneity by (NH4)2SO4, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5×10-10M. It was stable and active at pH 4-12 and also at 4-90°C for 1h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin. A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.

Purification, characterization, and antimicrobial activity of nontoxic trypsin inhibitor from Albizia amara Boiv.

The present investigation describes the purification, and characterization of a novel, thermostable, nontoxic, proteinaceous trypsin inhibitor extracted from seeds of Albizia amara Boiv. The proteinaceous protease inhibitor (API) was purified via acetone fractionation, ion-exchange chromatography (diethylaminoethyl (DEAE) cellulose), and gel permeation chromatography (GPC; Sephadex G-100). The apparent molecular weight of the API is 49kDa and is identified as a serine protease inhibitor. The API remains active in a wide pH range (3.0–8.0), and showed thermal stability at 60°C. The API inhibits trypsin by a mixed inhibitory mechanism with an inhibition constant (Ki) of 1.24×10−8M using BApNA (Nα-benzoyl-dl-arginine-p-nitroanilide hydrochloride) as the substrate. The API also showed substantial activity in the presence of several metal ions, surfactant (Triton X-100), oxidizing agent (dimethyl sulfoxide; DMSO), and NaCl (5%). Moreover, API retained 85% trypsin inhibition activity upon storage at 4°C over a period of 6 months. The antimicrobial effectiveness of the API against Pseudomonas aeruginosa, Bacillus subtilis, Alternaria alternata, Alternaria tenuissima, and Candida albicans reported in this study suggests its utility as a potential antimicrobial component.

Magnesium dependent proteinaceous protease inhibitor with pesticidal potential from alkali-halotolerant Streptomyces spp.: Optimization of production using statistical tools

Abstract The extensive diversity of microbial proteases in several physiological and industrial applications promoted search for newer protease inhibitors. Of 80 actinomycetes isolated from soil–water sediments of Soda lake on starch casein medium (1 M NaCl, pH 9.5) at 30 °C, only 5 have shown significant protease inhibitor (PI) activity (≃20 U) against trypsin. Based on the preliminary phenetic and 16S rRNA gene sequence, the closest related to VL 18, VL 50, VL 61 and VL J2 isolates was actinomycetes (99% identity). PI activity of culture broth of short listed strains ranged between 26.90 to 30.72 U. Alternative one variable assortments at a time (OVAT) for PI production by S treptomyces spp. VL J2 by optimized medium yielded 55.34 U within 96 h, the highest among the 5 isolates. Further, Plackett–Burman design (PBD) identified starch, KNO 3 and MgSO 4 for PI production by VL J2 strain as the most significant and they were further optimized by Response Surface Method (RSM) using a central composite design (CCD). Accordingly, 2.49% starch, 0.114% MgSO 4 and 0.249% KNO 3 were found to yield 72 U of PI by the Streptomyces sp . VL J2 in 96 h. The purified PI has molecular mass of ≃35 kD and showed inhibition of fungal mycelial growth and mid gut proteases of insect indicating the scope for its application as a bio control agent. The statistical tools employed in the present study showed 30% increase in PI production over OVAT approach and MgSO 4 was found to be a key factor in its production.

Ectopic expression of a Neospora caninum Kazal type inhibitor triggers developmental defects in Toxoplasma and Plasmodium.

Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.

Shape Complementarity in Serine Protease-inhibitor Complexes Correlate to Inhibition Constants

Natural proteinaceous protease inhibitors inhibit through nonproductive binding to proteases and steric blockage of active sites. These complexes are among the most structurally complementary protein-protein interactions. To see if complementarity is correlated to activity, we scored the shape complementarity in 15 serine protease-inhibitor complexes through in silico docking and compared the scores against their reported inhibition constants (Ki). A statistically significant, moderate and positive correlation was observed between shape complementarity and Ki (R = 0.58; P = 0.023). We also analyzed other physicochemical factors involved in serine protease-inhibitor interactions for correlation, but no other factor was correlated to Ki. However, significant correlations were observed between hydrogen bonds and interface areas (R = 0.762; P = 0.0004); and between hydrophobic interactions and free energies of solvation (R = -0.634; P = 0.011).

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea)

BackgroundProteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. MethodsPhylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. ResultsIn the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein–ligand and inter and intramolecular protein–protein hydrogen bonds. DiscussionThe genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea.

Rapid Detection of Proteinaceous Protease Inhibitor from Several Plants of North Maharashtra Region

Total 20 plants of North Maharashtra region were analysed for the presence of protease inhibitors in their seed. Protease inhibitors were detected from proteinaceous seed extracts by employing the rapid X-ray film method described in this investigation. Among studied plants, eleven plant seeds were reported to contain the protease inhibitor capable to inhibit various proteases like trypsin, pepsin, papain and metallo-protease. The testified results were authenticated by quantitative protease inhibition assay. The Albizia amara, Albizia lebbeck, Cassia australis, Mimosa hamata, Peltophorum pterocarpum , reported first time for the presence of trypsin inhibitor.

Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer

Proteinaceous protease inhibitors have potential application in medicines, agriculture and biotechnology. Present study was undertaken to purify and characterize a proteinaceous protease inhibitor from a medicinal plant, Senna tora syn. Cassia tora. The inhibitor was purified by ammonium sulphate precipitation, anion exchange (Q-sepharose), affinity (trypsin-sepharose) and molecular exclusion (sephadex G-75) chromatography. Zymography and denaturing polyacrylamide gel electrophoresis revealed a single band of ∼20kDa trypsin inhibitor. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix-assisted laser desorption ionization (MALDI) analyses revealed the presence of 19,725Da (pI 4.60) and ∼19,900Da (pI 4.57) isoform proteins in purified inhibitor. Protein identification by MALDI-peptide mass fingerprinting did not reveal high MASCOT (Matrix science) scores matching with previously known inhibitors. N-terminal amino acid sequence suggested this protein as a previously unreported inhibitor. Its dissociation constant (0.23×10−9M) was indicative of a high affinity trypsin inhibitor. The inhibitor was stable over a broad range of pH (4–10) and temperature (30–60°C). The purified inhibitor effectively inhibited total protease and trypsin-like activities of podborer (Helicoverpa armigera) midgut preparation. Hence, the inhibitor and its gene(s) can find application in combating against pest and protease dependent pathogens.

Proteinaceous Protease Inhibitor fromLawsonia Inermis: Purification, Characterization and Antibacterial Activity

A thermo-stable, proteinaceous protease inhibitor (LPI) from Lawsonia inermis is reported. The LPI was purified from Lawsonia inermis seeds by subsequent ammonium sulfate precipitation, ion exchange chromatography (DEAE-Cellulose) and gel permeation chromatography (Sephadex-50). The purified protease inhibitor is effective against a wide range of proteases viz. papain, trypsin, pepsin and metallo-protease. The apparent molecular weight of the protease inhibitor is 19 kDa, determined by SDS-PAGE electrophoresis. The protease inhibitor was found to be stable at 70 degrees C for 30 min. It was also examined for antibacterial activity against Pseudomonas aeruginosa MTCC 7926 and Staphylococcus aureus NCIM 2079; the IC50 values of the purified LPI were 11.4 microg/mL and 16.6 microg/mL respectively.

Screening of Phytochemical Compounds and Toxic Proteinaceous Protease Inhibitor in Some Lesser-known Food Based Plants and Their Effects and Potential Applications in Food

Compounds or substances which act to reduce nutrient intake, digestion, absorption and utilization and may produce other adverse effects are referred to as anti-nutrients or anti-nutritional factors. Plant sources contain in their raw state wide varieties of anti-nutrients which are potentially toxic. This study was conducted to phytochemical investigation on fruits and plants from India and Iran. Eight plant species were used. A qualitative phytochemical analysis was performed for the presence of polyphenols, saponins, alkaloids, phytic acid, trypsin inhibitor and saponins<b>. </b>The results obtained of the investigated plants showed that the all eight samples were found to be polyphenols but <i>Solanum</i> <i>indicum</i> contains highest value(7.02mg/g). All samples showed the presence of alkaloids, and showed presence of saponins except <i>A</i><i>locacia</i> and <i>A</i><i>s</i><i>paragus</i> and showed presence of steroids except <i>C</i><i>hloroph</i><i>ytum </i>and <i>A</i><i>sparagus</i>. <i>Portulaca</i> with(16.9 TIU/g) and <i>A</i><i>sparagus </i>with(0.8 TIU/g) contain highest and lowest values of trypsin inhibitor respectively. The phytochemical screening revealed moderate phytate contents except solanum and portulaca. The plants are rich sources of polyphenols appear to have protective effects for human health. Trypsin inhibitor and phytic acid may play a role as an antioxidant in plants and may be beneficial to health when it is consumed.

Screening of Phytochemical Compounds and Toxic Proteinaceous Protease Inhibitor in Some Lesser-Known Food Based Plants and Their Effects and Potential Applications in Food

Compounds or substances which act to reduce nutrient intake, digestion, absorption and utilization and may produce other adverse effects are referred to as anti-nutrients or anti-nutritional factors. Plant sources contain in their raw state wide varieties of anti-nutrients which are potentially toxic. This study was conducted to phytochemical investigation on fruits and plants from India and Iran. Eight plant species were used. A qualitative phytochemical analysis was performed for the presence of polyphenols, saponins, alkaloids, phytic acid, trypsin inhibitor and saponins. The results obtained of the investigated plants showed that the all eight samples were found to be polyphenols but Solanum indicum contains highest value(7.02mg/g). All samples showed the presence of alkaloids, and showed presence of saponins except Alocacia and As- paragus and showed presence of steroids except Chlorophytum and Asparagus. Portulaca with(16.9 TIU/g) and Asparagus with(0.8 TIU/g) contain highest and lowest values of trypsin inhibitor respectively. The phytochemical screening revealed moderate phytate contents except Solanum and Portulaca. The plants are rich sources of polyphenols appear to have protec- tive effects for human health. Trypsin inhibitor and phytic acid may play a role as an antioxidant in plants and may be beneficial to health when it is consumed.

Antiviral Potential of a Proteolytic Inhibitor from Streptomyces Chromofuscus 34–1

ABSTRACTThe novel proteinaceous protease inhibitor, isolated from the culture supernatants of Streptomyces chromopfuscus 34–1 (SS 34–1) demonstrated a specific and selective anti-influenza virus effect. By the use of complementary virological assays it was demonstrated that the expression of the viral haemagglutinin (HA) on the surface of infected cells, the virus-induced cytopathic effect (CPE) and the infectious virus yields (IVY), used as measures of A/Germany/34, strain Rostock (H7N1) (A/Rostock) virus growth, were all reduced at non-toxic concentrations of SS 34–1 in a dose-dependent way. In a single cycle of viral growth, the penetration of viral particles and the late stages of viral replication were the most susceptible to inhibition.The preparation protected mice from mortality in the experimental A/Aichi/2/68 (H3N2) influenza virus infection. The SS 34–1-treatment (0.16 mg/mouse/day) of virus-infected mice led to a considerable reduction of mortality rate (index ofprotection = 71.0 %) and marked prolongation of mean survival time (+ 5.5 days). The lesions of the lungs due to infection as well as lung infectious virus titres were significantly reduced—δ log10 TCID50/ml up to 4.5. The application of SS 34–1 induced a continuous rise of the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. The infection triggered a slight reduction of protease activity in the lungs at 5 and 48 h p.i. and a marked increase at 24 h and 6 day p.i. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h, 6 and 9 day p.i. SS 34–1-treatment brought both activities to normal levels. It was assumed that SS 34–1 exerted its inhibitory effect indirectly, through an increase of the protease-inhibitory activity. Histopathological examination of the lungs showed that under SS 34–1-treatment pulmonary lesions were less severe than those of untreated controls.The isolated novel protease inhibitor was purified by anion-exchange chromatography and reversed phase-HPLC. Analysis by differential scanning calorimetry (DSC) revealed that at pH 7.5 the denaturation temperature was 75 °C and the denaturation itself was irreversible. Analysis by circular dichroism (CD) in the UV region 190–250 nm showed that at pH 7.5 the spectrum presented 2 minima at 208 and 222 nm, typical for an α-helical structure. At pH 2.5 and after heating the spectrum corresponded to an unordered structure.

A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract.

BackgroundProteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases.MethodsThe crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools.ResultsThe crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD) of protease inhibitor (PI) was detected in dialyzed and SEC elute. The conidial germination of Aspergillus flavus was moderately inhibited (30%) by the dialyzed seed extract.ConclusionsCassia tora seed extract has strong protease inhibitory activity against trypsin, Aspergillus flavus and Bacillus sp. proteases. The inhibitor in Cassia tora may attenuate microbial proteases and also might be used as phytoprotecting agent.