Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI‐induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI‐induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.     

Inhibition of digestive trypsins by plant Kunitz proteins reduces the viability of Spodoptera cosmioides larvae

The acquired resistance by insects and the harmful environmental effects of chemical pesticides have encouraged the search of new tools for proper pest management. Among them, the use of protease inhibitors (PIs) obtained from plants has gained interest because they are a natural system against herbivory, are organic molecules with higher specificity and have the potential to cause less damage to nature. The aim of this work was to characterise the inhibitory potential of the proteins ApTI (Adenanthera pavonina trypsin inhibitor) and ILGA (Inga laurina trypsin inhibitor) on the digestive trypsins of Spodoptera cosmioides through molecular docking, enzymatic kinetics and biological survival analyses. The docking between trypsins and inhibitors was performed using the program CLUSPRO; the inhibitory constant Ki and the inhibition type were determined through chromogenic assays. In order to analyse survival, several concentrations of ApTI and ILTI inhibitors were included in the artificial diet of neonatal larvae. In this study, we determined that ILTI binds to the active site of the trypsins with a specificity similar to its natural substrate, whereas APTI showed that the inhibitor reactive site is not in contact with the trypsins catalytic site. The ILTI and APTI inhibitors were characterized as competitive and uncompetitive tight‐binding inhibitors, respectively. The survival curves obtained using Kaplan–Meier estimators indicated that the lowest percentage survival (20%) for all inhibitors tested was obtained using 1.0% doses at a development time of less than 20 days. We concluded that ILTI and APTI present biotechnological potential as agents against phytophagous Lepidoptera insects, inhibiting trypsins through tight‐binding inhibition, with competitive and non‐competitive mechanisms, respectively. The effect of ApTI and ILTI on the development of S. cosmioides larvae is shown to be toxic.     

Anionic Trypsin from North Pacific Krill (<Emphasis Type="Italic">Euphausia pacifica</Emphasis>): Purification and Characterization

An anionic trypsin (TRY-EP) was purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzyme was identified as a trypsin by LC-ESI-MS/MS analysis. The relative molecular mass of TRY-EP was 33 kDa, with isoelectric point of 4.5. The histidine, tryptophan, arginine, lysine, aspartic acid and glutamic acid residues were functional groups to TRY-EP. TRY-EP was activated by Ca²⁺ and Mg²⁺ and inhibited by some heavy metal ions (Zn²⁺, Cu²⁺, Pb²⁺ and Hg²⁺), organic solvents (ethanol, glycerin, DMSO and acetone) and specific trypsin inhibitors (benzamidine, CEOM, SBTI and TLCK). TRY-EP was active over a wide pH (6.0–11.0) and temperature (10–70°C) range, with optimum of pH 9.0 and 40–50°C. TRY-EP was stable between pH 6.0 and 11.0 and below 30°C. Compared with some trypsins from the Temperate and Tropical Zone organisms, TRY-EP and other trypsins from the Frigid Zone organisms have higher affinity to substrate and 2–42-fold physiological efficiency.     

A Trypsin Inhibitor from <Emphasis Type="Italic">Sapindus saponaria</Emphasis> L. Seeds: Purification,Characterization, and Activity Towards Pest Insect Digestive Enzyme

The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS–PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.     

BIOPOTENCY OF SERINE PROTEASE INHIBITORS FROM COWPEA (Vigna unguiculata) SEEDS ON DIGESTIVE PROTEASES AND THE DEVELOPMENT OF Spodoptera littoralis (BOISDUVAL)

Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE‐Sephadex A‐25 column. Cream7‐purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (K_i) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect‐resistant transgenic plants.     

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protect Acacia food bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two‐dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz‐type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin‐like and elastase‐like proteolytic activity in the guts of seed‐feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase‐like and to a lower degree chymotrypsin 1‐like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term ‘exclusive rewards’ can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.     

Solar UV‐B radiation and ethylene play a key role in modulating effective defenses against Anticarsia gemmatalis larvae in field‐grown soybean

Solar UV‐B radiation has been reported to enhance plant defenses against herbivore insects in many species. However, the mechanism and traits involved in the UV‐B mediated increment of plant resistance are unknown in crops species, such as soybean. Here, we studied defense‐related responses in undamaged and Anticarsia gemmatalis larvae‐damaged leaves of two soybean cultivars grown under attenuated or full solar UV‐B radiation. We determined changes in jasmonates, ethylene (ET), salicylic acid, trypsin protease inhibitor activity, flavonoids, and mRNA expression of genes related with defenses. ET emission induced by Anticarsia gemmatalis damage was synergistically increased in plants grown under solar UV‐B radiation and was positively correlated with malonyl genistin concentration, trypsin proteinase inhibitor activity and expression of IFS2, and the pathogenesis protein PR2, while was negatively correlated with leaf consumption. The precursor of ET, aminocyclopropane‐carboxylic acid, applied exogenously to soybean was sufficient to strongly induce leaf isoflavonoids. Our results showed that in field‐grown soybean isoflavonoids were regulated by both herbivory and solar UV‐B inducible ET, whereas flavonols were regulated by solar UV‐B radiation only and not by herbivory or ET. Our study suggests that, although ET can modulate UV‐B‐mediated priming of inducible plant defenses, some plant defenses, such as isoflavonoids, are regulated by ET alone.     

Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna

Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient‐rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin‐inhibitor‐containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC₅₀ values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour‐joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.     

Purification and some properties of two anionic trypsins from the eel (Anguilla japonica)

Two anionic enzymes, designated as trypsins 1 and 2, were purified from the pancreas of the eel Anguilla japonica by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The final preparation of trypsin 1 was homogeneous but that of trypsin 2 still contained impurities. Both enzymes had similar pH optima of near 8.3 for the hydrolysis of N-tosyl-L-arginine methyl ester. Trypsin 1 was stabilized by calcium ions but the stability of trypsin 2 was not affected by calcium ions. Both enzymes were inhibited by typical trypsin inhibitors including serine proteinase inhibitors.     

Activities of anionic and cationic trypsins in the temperature range from 5 to 37 degrees C. Mutant anionic trypsins as a model of cold-adapted (psychrophilic) enzymes

Temperature dependences of kinetic constants (k _cat and K _m) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of coldadapted fishes. The anionic and cationic trypsins were found to differ in organization of the S₁ -substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S₁) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K _m value) to the anionic and cationic trypsins and on the catalytic activity k _cat should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.     

Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus

1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH4)2SO4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. Both trypsins catalyzed the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. The enzymes had a pH optimum of 8-9 for the hydrolysis of BAPNA and of 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. Trypsins A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca2+ for activity and stability.     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

Cold Adaptation,Ca2+ Dependency and Autolytic Stability Are Related Features in a Highly Active Cold-Adapted Trypsin Resistant to Autoproteolysis Engineered for Biotechnological Applications

Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca²⁺ dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10^° C and at least 50% more active than pig trypsin up to 50^° C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca²⁺ concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca²⁺ dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.     

Coevolution of insect trypsins and inhibitors

Many plant proteinase inhibitors have lysine at the P1 position, presumably to avoid hydrolysis by insect trypsins. Lepidopteran trypsins appear to have adapted to resist proteinase inhibitors through increased inhibitor hydrolysis and decreased binding to inhibitor hydrophilic reactive sites. Lepidopteran digestive trypsins prefer lysine at the P1 position and have substrate binding subsites more hydrophobic than trypsins from insects in other orders. All available sequences of sensitive and inhibitor-insensitive insect trypsins were aligned with porcine trypsin, for which interactions with Kunitz and Bowman-Birk inhibitor are known. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the following residues were considered important to insect trypsin-PI interactions (chymotrypsin numbering): 60, 94, 97, 98, 99, 188, 190, 213, 215, 217, 219, 228. These residues support the Neighbor Joining analysis tree branches separating sensitive and insensitive trypsin sequences. Primary sequences interacting with PIs are around the active site, with some forming part of the S1 (188, 217, 219 and 228) or S4 (99, 215) pockets.     

Biochemical characterization and structural analysis of trypsin from Plodia interpunctella midgut: implication of determinants in extremely alkaline pH activity profile

In the present study, trypsin from Plodia interpunctella (Hübner) is characterized to discover sequence, biochemical and structural features. This enzyme is purified by ion exchange chromatography using fast protein liquid chromatography on proteins from fifth‐instar larvae. The enzyme is optimally active at 50 °C and pH 11.0. The kinetic parameters (K_m and V_max) of the enzyme are 5.3 ± 0.6 µm and 31 ± 1.3 nmol min^?1 mg^?1, respectively (using Nα‐benzoyl‐l ‐arginine ρ‐nitroanilide hydrochloride as substrate). The enzyme is inhibited by the addition of Cu²⁺ and Mn²⁺, whereas it is activated by Li⁺ at high concentrations. Moreover, the enzyme is almost completely inhibited in the presence of Nα‐tosyl‐l ‐lysine chloromethyl ketone hydrochloride and phenylmethanesulphonyl fluoride. To understand some characteristics of P. interpunctella trypsin, including active site structure and alkaline pH profile, a reliable structural model of P. interpunctella trypsin is built based on the Fusarium oxisporum (Schlecht) trypsin cystal structure (Protein Data Bank code: 1GDU). The secondary structure content of the purified trypsin from near‐ultraviolet circular dichroism data shows considerable similarities with that of P. interpunctella trypsin predicted structure. Analysis of pK_a values of active site residues, a type of amino acid residue in the active site cleft and the surface charges of the model and Tribolium castaneum (Herbst) trypsin structure as an insect species from different orders reveals some differences between them. These differences might effect on the microenvironment of the active site cleft and consequently shift its pH profile. The application of multiple theoretical and experimental techniques is well adapted to predict the enzyme structure with high accuracy and this could help in the design of a powerful inhibitor for trypsin with ideal properties.     

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications

We characterized trypsin‐ and chymotrypsin‐like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio‐formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76‐ and 14.05‐fold for trypsin and chymotrypsin, respectively. Michaelis–Menten constants (K_m) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ～21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ～29 and 34 kDa in SDS‐PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio‐formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.     

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

A TRYPSIN‐LIKE PROTEINASE IN THE MIDGUT OF Ectomyelois ceratoniae ZELLER (LEPIDOPTERA: PYRALIDAE): PURIFICATION,CHARACTERIZATION, AND HOST PLANT INHIBITORS

A trypsin‐like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G‐100 and DEAE‐cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh‐Sabz, May‐Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17–20 of Brait; fractions 18 and 21–26 of Torsh‐Sabz; fractions 1–7, 11–17, and 19–21 of May‐Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May‐khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.     

The isolation and partial characterization of trypsin from the pancreas of the ostrich Struthio camelus

Cationic trypsin was isolated and purified from the pancreas of the ostrich (Struthio camelus) by affinity chromatography on a Trasylol-Sepharose column. External activation of trypsinogen was required before trypsin could be isolated. The final preparation was homogeneous by SDS-PAGE and by sedimentation equilibrium centrifugation studies, resulting in Mr values of 24,547 and 22,091, respectively. The Mmin value obtained from amino acid analysis was 22,450. A mean sedimentation coefficient of 2S was obtained by sedimentation velocity centrifugation. Results obtained from N-terminal and amino acid analyses were similar to those from trypsins of other species. The effects of pH, temperature and inhibitors (LBTI, KBPTI and PMSF) on the tryptic activity were examined. The effect of calcium ions and enzyme concentration on the rate of self-digestion of ostrich trypsin was also investigated.