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.     

A functional comparison of ovine and porcine trypsins

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.     

Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix)

Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 °C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 °C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl₂ partially protected trypsin against activity loss at 40 °C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.     

Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus)

A cationic trypsin (trypsin A) and an anionic trypsin (trypsin B) were highly purified from the hepatopancreas of the Japanese sea bass (Lateolabrax japonicus) by ammonium sulfate precipitation, column chromatographies of DEAE-Sepharose and Sephacryl S-200 HR. Purified trypsins revealed single band on SDS-PAGE and their molecular masses were 21 kDa and 21.5 kDa, respectively. Trypsins A and B exhibited maximal activity at 40°C, and shared the same optimal pH at 9.0 using Boc-Phe-Ser-Arg-MCA as the substrate. The two trypsins were stable up to 45°C and in the pH range from 7.0 to 11.0. Trypsin inhibitors such as Pefabloc SC, PMSF and benzamidine are effective to these two enzymes and their susceptibilities were similar. Apparent K(m)s of trypsins A and B were 1.12 and 0.7 μM and k(cat)s of them were 72.08 and 67.79 S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. The N-terminal amino acid sequences of the two trypsins were determined to the 24th residues, which were highly identical to trypsins from other species of fish while trypsins A and B only shared 45.8% identity. The digestive effect of the two trypsins on native shrimp muscular proteins indicated their effectiveness in the degradation of food proteins.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

大鼠、小鼠胰蛋白酶的分离纯化及动力学性质

采用STI-Sepharose 4B亲和层析的方法,从鼠新鲜胰脏中分离得到纯的胰蛋白酶。大鼠胰蛋白酶的比活为24 615BAEEU/mg蛋白,总活性回收率47％,小鼠胰蛋白酶的比活为32 768BAEEU/mg蛋白,总活性回收率55％。经SDS-聚丙烯酰胺凝胶电泳鉴定,大鼠、小鼠胰蛋白酶均呈现单一蛋白带,两者的分子量都是24kD。用等电聚焦电泳测定,二者的等电点均为p19.5以上。对它们的动力学性质作了研究,大鼠胰蛋白酶的Km值为2.33×10~(-4)mol/L,K,值为0.92×10~(-5)mol/L,小鼠胰蛋白酶的Km值为5.60×10~(-4)mol/L,K:值为1.27×10~(-5)mol/L。     

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.     

赤豆子叶β-半乳糖苷酶的纯化及其性质的研究

β-半乳糖苷酶 ( EC3.2 .1 .2 )广泛存在于动植物的组织中 ,如在杏仁、桃子、大豆、咖啡豆等植物 ,蜗牛 ,哺乳动物的肠道中都有 β-半乳糖苷酶 .同样 ,微生物也能产生β-半乳糖苷酶 ,俗称乳糖酶 .乳糖操纵子学说的提出就是建立在对微生物β-半乳糖苷酶研究基础之上的 .在过去的研究中 ,关于微生物、动物来源的乳糖酶报道较多[1] ,而对于植物来源的β-半乳糖苷酶研究报道却相对较少[2 ] .它可能降解多糖中 β-构型半乳糖苷键 ,为种子生长发育提供必要的能量来源 .但目前对β-半乳糖苷酶在植物中确切的生理生化功能尚不清楚 .为了进一步阐明…     

Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.     

Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.     

Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum

Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S^?1, respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization.     

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.     

Towards genetic based insect resistance in strawberry using the Cowpea trypsin inhibitor gene

An efficient shoot regeneration system has been developed involving organo-genesis from stem tissue of the strawberry cultivars Melody, Rhapsody and Symphony. Regeneration also occurred in the presence of antibiotics. The system was shown to be suitable for transformation using the GUS:Intron marker gene construct. Histological analysis using the substrate 5-bromo-4-chloro-3-indolyl B-D-glucuronide (X-Gluc) and the polymerase chain reaction (PCR) using specific primers permitted the identification of transformants.
Inoculations were then carried out using the Cowpea protease trypsin inhibitor (CpTi) gene construct. A simple visual assay technique was developed to identify those plants expressing CpTi, allowing them to be selected for further analysis. Trypsin acts on the substrate α-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) to produce p-nitroaniline (p-na). Trypsin inhibition CpTi was easily detected as a reduction in or elimination of yellow colour development which occurred as the hydrolysis of BAPNA to pna proceeded (Preiser, Schmitz, Maestracci & Crane, 1975). The polymerase chain reaction (PCR) was used to confirm the presence of the gene sequence.     

Purification and characterization of trypsin-like enzymes from North Pacific krill (<Emphasis Type="Italic">Euphausia pacifica</Emphasis>)

Two trypsin-like enzymes (TLEs) were purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzymes were identified as trypsins by LC-ESI-MS/MS analysis. The relative molecular mass of TLE I and TLE II were 33 and 32.3 kDa, respectively, with isoelectric points of 4.5 and 4.3, respectively. The TLEs showed excellent thermal stable in the crude extract and the purified TLEs were active over a wide pH (6.0–11.0) and temperature (10–70°C) range. Compared with trypsins from other organisms, the purified TLEs had physiological efficiencies of 1.6–6.7-fold. The difference in Arg, Ile and Asp content might explain why E. pacifica TLEs have good thermal stability and physiological efficiency.     

Hydrolysis by Indigenous Plasmin: Consequences for Enzymatic Cross-Linking and Acid-Induced Gel Formation of Non-Micellar Casein

Casein is a group of milk proteins with high nutritional value, and the exploitation of its techno-functional potentials has been investigated for decades. In this study, acid casein powder was dissolved in 0.1 mol/L phosphate buffers with different pH, resulting in casein solutions with pH 5.9, 6.6 and 7.3. During preparation and storage (40 °C) of the samples, casein hydrolysis was observed in size exclusion chromatography and gel electrophoresis. The degree of hydrolysis increased with increasing pH, and treatment of casein with commercial plasmin resulted in similar polypeptides, suggesting that the hydrolysis was caused by residual indigenous plasmin present in the acid casein powder. Most polypeptides could be cross-linked by microbial transglutaminase, except for one particular fraction which appeared at constant intensity in the chromatograms. The stiffness of acid-induced gels as determined in small amplitude oscillatory shear rheology decreased with increasing degree of hydrolysis, and was also lower for cross-linked samples when the preceding casein hydrolysis was more pronounced. Enzymatic cross-linking increased the resistance of casein against plasmin-related hydrolysis, presumably because of the resulting lysine modification. However, one particular fraction of polypeptides was released by hydrolysis in spite of cross-linking, suggesting that they did not contain lysine residues that are susceptible for mTGase. The results indicate that plasmin-related hydrolysis should be taken into account for the application of acid casein or sodium caseinate as additive in food design.

     

Hydrophobic-ionic chromatography. Its application to purification of porcine pancreas enzymes.

1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 AND 5.3) WITH 0.5 M acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 M 3,3-dimethyl glutarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of the pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fmately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases. The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.07 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.     

Separation of a bradykinin-releasing enzyme from the proteolytic complex of Levantine viper venom

Two serine hydrolases have been separated from the proteolytic complex of the venom of Levantine viper, Vipera lebetina turanica. The enzyme with mol. weight of 50,000 +/- 5,000, pH-optimum of 8.5 and isoelectric point in the range of 5.6--6.6 had proteolytic activity against casein and hydrolyzed benzoyl-arginine p-nitroanilide. The other enzyme with mol. weight of 37,000 +/- 2,000, pH optimum of 9 and isoelectric point in the range of 4.1--4.5 had no effect on benzoylarginine p-nitroanilide, casein or hemoglobin, but possessed a bradykinin-releasing activity. Both enzymes were stereoselective against L-arginine, hydrolyzing tosyl-L-arginine methyl ester without having any effect on D-arginine ester. The interaction of the enzymes with a number of N(alpha)-arylsulfonylarginine methyl esters has been studied. The influence of the substitute X in the arylsulfonyl part of the substrates upon their hydrolysis by the bradykinin-releasing enzyme has been described by the Hammett equation of rho omicron = 1.14 +/- 0.33 (r = 0.974).     

Purification and partial characterization of aminopeptidase A from the serum of pregnant and non-pregnant women

Aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7) was purified from human maternal and control sera using CM-cellulose chromatography, DEAE-Sephacel chromatography, Sephacryl S-300 gel filtration and hydroxyapatite chromatography. The purification coefficients were 3069 and 5210 and the yields 6.3 and 6.1% for the maternal and control serum, respectively. The purified enzymes appeared free from other serum aminopeptidases in polyacrylamide gel electrophoresis. The biochemical and physical characteristics of the enzymes from maternal and control sera were similar. A molecular weight of 260 000, an optimum at pH 6.75-7.25 and a fairly good stability of the enzymes at 4 and -18 degrees C were recorded. The alkaline earth metals (Ca2+, Ba2+, Sr2+) were the activators of alpha-L-glutamyl-beta-naphthylamide hydrolysis, while alpha-L-aspartyl-beta-naphthylamide hydrolysis was markedly potentiated with Ca2+ but not with Ba2+ at all. The most rapid hydrolysis was shown with GluNA (Km with Ba2+ 0.156 +/- 0.014 mM and 0.136 +/- 0.009 mM in maternal and control serum, respectively), while only minimal hydrolysis of some neutral and basic amino-acid-beta-naphthylamides were observed. The contribution of the placenta to the elevated aminopeptidase A levels in the pregnancy plasma could not be solved on the basis of the present observations.     

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.     

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.