Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Characterization of α-amylase inhibitor from rice bean with inhibitory activity against midgut α-amylases from Spodoptera litura

The α-amylase inhibitor (α-AI) activity varied from 7.529 to 10.766 (IU/g) in 13 rice bean with different genotypes. BRS-2 exhibited the highest α-AI activity (55.3%). Rice bean α-AI was purified to homogeneity by 80% ammonium sulfate precipitation, dialysis, ion exchange chromatography on DEAE-Sepharose and gel filtration through Superdex-75. Its homogeneity was confirmed by SDS-PAGE under reducing conditions showing a single band protein of molecular weight 25 kDa. The inhibitor was purified to 75.9 fold with final yield of 28.0% with specific activity of 660.2 IU. Inhibition studies carried out at pH from 2.2 to 9.0 revealed pH optimum at pH 6.9 (69.3%). The maximum α-AI activity was found at 37°C (68.8 %) and the lowest was revealed at 100°C (37.0%). Optimum inhibitory activity was expressed during pre-incubation of enzyme with inhibitor at pH 6.9 and 37°C. Isoelectric focusing of purified inhibitor showed a single band near pH 4.7. The first 6 amino acids in the N-terminus were recorded as Ala-Ser-Ser-Arg-Phe-Cys (ASSRFC). The purified inhibitor inhibited the α-amylase from the larval midgut of Spodoptera litura up to 86.6%. The α-amylase inhibitors are important seed storage proteins because of their potentiality for exploitation in pest control and crop defense against insect infestation. Their expression at high levels can confer resistance in transgenic legumes, which could be exploited for crop improvement.     

Studies on Trypsin Inhibitor in Bean (Phaseolus vulgaris L) Cultivars of Himalayan Region

Eight Phaseolus vulgaris L cultivars of Himalayan region were analyzed for trypsin inhibitor activity and inhibition of gut trypsin enzyme extracted from Spodoptera littoralis larvae. Trypsin unit inhibited per gram seed weight was maximum in local yellow cultivar. The trypsin inhibitor was purified to 65.9-fold with 55.6% recovery from seeds of selected cultivar. The purified protein had a molecular weight of 14,130 Daltons and was found to be a monomer by SIDS-PAGE. It was heat stable at 100°C for 10 minutes and had a pH optimum of 7.5. Hence, the purified inhibitor appears to be of Bowman-Birk type. It lost its activity on exposure to 0.2M 2-mercaptoethanol. The inhibition pattern was of non-competitive type and the Ki value was 0.8μM. The KM value of trypsin enzyme for the substrate BApMA was 2.2mM.     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.     

Inhibition of midgut protease of yellow stem borer (<Emphasis Type="Italic">Scirpophaga incertulas</Emphasis>) by cysteine protease-like inhibitor from mature jackfruit (<Emphasis Type="Italic">Artocarpus heterophyllus</Emphasis>) seed

A novel protease inhibitor was isolated and purified from the mature seeds of jackfruit (Artocarpus heterophyllus) by precipitation with ammonium sulphate, followed by DEAE-cellulose and gel filtration (Sephadex G-100) chromatography. The isolated protease inhibitor strongly inhibited papain and midgut proteases of yellow stem borer (Scipophaga incertulas) larvae, as seen by in vitro assay. The purified protease inhibitor was active over a wide range of pH with the maximum activity between pH 4 and 10. This protein was also stable up to 80°C, but the retained activity was lost at 100°C, when heated for 30 min. The molecular mass of the purified cysteine-like protease inhibitor is to be 14.50 kDa as determined by SDS-PAGE. Significant reduction in larval weight and mortality was observed, when fresh rice culms with protease inhibitor was feeded to the yellow stem borer larvae. These results may provide important information to control the yellow stem borer in rice with respect to naturally occurring insecticidal proteins. The observed differences would potentially translate into reductions in population growth of yellow stem borer, indicating a potential value of using jackfruit protease inhibitor for protecting rice plants against damage by the yellow stem borer.     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Enzymatic Properties of Purified Alkaline Proteinase from Aspergillus sojae†

Some enzymatic properties of purified alkaline proteinase from Aspergillus sojae were investigated. The optimum pH for casein digestion was 11.0. The enzyme activity was almost completely lost at 60°C within ten minutes. At low temperature, the enzyme was highly stable at the range of pH 4.5 to 10.0. At 50°C, the most stable pH was around 6.0. None of metallic ions tested promoted the activity, but Hg²⁺ showed a remarkable inhibition. The Hg²⁺-treatment seemed to cause a large unfolding of the enzyme molecule. The enzyme was inhibited by potato inhibitor and a number of animal sera. Metal chelating reagents and sulfhydryl reagents tested had no effect on the activity, but DFP caused a marked inhibition. The sensitivity to DFP of the enzyme was about 1/300 of that of α-chymotrypsin. The enzyme was inhibited neither by TPCK nor by TLCK. As the result it was assumed that the structure of the active site of the enzyme is fairly different from that of trypsin, or of chymotrypsin.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Physicochemical Properties of Niigata Waxy Rices

The crude lipase powder has been purified 216-fold in specific activity by means of pH adjustment, DEAE-Cellu1ose, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 column chromatography and the recovery of the activity was 30%. The purified lipase was confirmed to be homogeneous with disc electrophoresis and ultracentrifugal analyses. The purified lipase was stable in the pH range from 7.0 to 10.0. Optimal pH for the lipolysis of polyvinyl alcohol-emulsified olive oil at 45°C was 8.0 and optimal temperature was 60°C. The purified lipase was stable up to 60°C and retained 55% of full activity after heating at 70°C for 20 min.     

A kidney bean trypsin inhibitor with an insecticidal potential against Helicoverpa armigera and Spodoptera litura

In the present study, trypsin inhibitor extracts of ten kidney bean seed (Phaseolus vulgaris) varieties exhibiting trypsin and gut trypsin-like protease inhibitor activity were tested on Helicoverpa armigera and Spodoptera litura. Trypsin inhibitor protein was isolated and purified using multi-step strategy with a recovery of ~15 % and purification fold by ~39.4. SDS-PAGE revealed a single band corresponding to molecular mass of ~15 kDa and inhibitory activity was confirmed by reverse zymogram analyses. The inhibitor retained its inhibitory activity over a broad range of pH (3–11), temperature (40–60 °C) and thermostability was promoted by casein, CaCl₂, BSA and sucrose. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.85 μM. The purified trypsin inhibitor protein was further incorporated in the artificial diet and fed to second instar larvae. A maximum of 91.7 % inhibition was obtained in H. armigera, while it was moderate in S. litura (29 %) with slight varietal differences. The insect bioassay showed 40 and 22 % decrease in larval growth followed by 3 and 2 days delay in pupation of H. armigera and S. litura, respectively. Some of the adults emerged were deformed and not fully formed. Trypsin inhibitor protein was more effective against H. armigera as it showed 46.7 % mortality during larval growth period compared to S. litura (13.3 %).     

An acid stable trypsin-chymotrypsin inhibitor from horse gram (Dolichos biflorus)

An inhibitor of trypsin and chymotrypsin was purified from horse gram (Dolichos biflorus) beans. The concentration of the inhibitor which provided total inhibition was 0.27 Μg/Μg tryptic enzyme and 0.46 Μg/Μg chymotryptic enzyme. The inhibitor was stable at 37‡C between pH of 3 to 11 and at 97‡C, upto pH 5.0 only. While the activities were rapidly lost in 0.1N NaO H the loss was only 5 0% in 0.1N HCl when kept for 2 h at 97‡C. On heating at pH 7.8, it remained stable upto 80‡C with a gradual loss in activities at 97‡C and a total loss occurring by autoclaving at 15 psi for 10 min. Reduction of disulphide bonds by 2-mercapto-ethanol, pronase digestion and boiling in the presence of 1 M NaCl led to reduction in the activities. However, the inhibitor was resistant to the action of pepsin and subtilisin and to urea at 37‡C.     

Longevity and germination of Edhazardia aedis (Microspora: Amblyosporidae) spores

Edhazardia aedis is a polymorphic microsporidium of mosquitoes that is both horizontally and vertically transmitted to its host. Because it is being developed for biological control of mosquitoes, detailed knowledge is needed regarding the longevity and germation of its fragile, mosquito‐infectious spore. Spores responsible for horizontal transmission were extracted from 7–10‐day‐old larvae (reared from infected Aedes aegypti eggs) and purified by Ludox density gradient centrifugation. Mature spores were variable in specific gravity, being found throughout the 20 and 60% zone in Ludox gradients. The optimal environment for spore germination was dilute KCl at pH 10.0–11.0; ammonia inhibited germination. Osmotic inhibition was almost complete in both sucrose and polyethylene glycol at concentrations equivalent to 40 atm. The spores retained their viability for a maximum of 21 days at 23±2°C, whereas when held at 5±2°C, their viability was completely lost within two days post‐harvest. Potential for germination decreased along with infectivity, providing a simple assay for spore viability.     

Enzymatic Properties of Rice Bran Lipase

The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 37°C. It was stable over the pH range from 4 to 9 and below 40°C, but completely lost the activity by heating 60°C for 15 min. The enzyme was activated by low concentration of calcium ion but inhibited partially by high concentration of calcium ion and by EDTA. With respect to substrate specificity, the enzyme exhibited a high specificity toward triglycerides and hydrolyzed ester bonds of short-carbon chain triglycerides faster than long-carbon chain triglycerides, whereas it catalyzed the hydrolysis of the oils from rice bran, olive and coconut. When 2-oleo-1,3-distearin was used as substrate, the enzyme was capable of preferentially hydrolyzing fatty acid ester bonds at the 1,3-position.     

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.     

Effects of temperature on the cell wall and osmotic properties in dark-grown rice and azuki bean seedlings

The mechanism inducing the difference in growth rate under various temperature (10–50 °C) conditions was analyzed using rice and azuki bean seedlings. The growth rate of rice coleoptiles and azuki bean epicotyls increased as temperature increased up to 40 and 30 °C, respectively, and the elongation was retarded at a higher temperature. The cell wall extensibility of rice coleoptiles and azuki bean epicotyls also showed the highest value at 40 and 30 °C, respectively, and became smaller as the temperature rose or dropped from the optimum. The opposite tendency was observed in the minimum stress-relaxation time of the cell wall. On the other hand, the cellular osmotic concentration of rice coleoptiles and azuki bean epicotyls was lower at the temperature optimum for growth at 40 and 30 °C, respectively. When rice and azuki bean seedlings grown at 10, 20, 40, or 50 °C were transferred to the initial temperature (30 °C), the growth rate of coleoptiles and epicotyls was mostly elevated, concomitant with an increase in the cell wall extensibility. The growth rate was correlated with the cell wall mechanical parameters in both materials. These results suggest that the environmental temperature modulates the growth rate of plant shoots by affecting mainly the mechanical properties of the cell wall. Electronic Publication     

Studies on the Autolysis of Aspergillus oryzae

Properties of nuclease O, a new intracellular enzyme which was partially purified from autolyzate of Asp. Oryzae,¹⁾ are described in this paper. The purified enzyme preferentially depolymerized RNA and heat denatured DNA, but apparently did not attack native DNA. It was activated by 0.1 mm Mg²⁺ or Mn²⁺, and inactive in the presence of EDTA. Optimum pH of the activity were 7.7 for DNA and 8.2 for RNA. By heat treatment (60°C, 10 min at pH 6) the nuclease completely lost its activity for RNA and DNA. Optimum concentration of Tris buffer for enzymatic activity was 0.15~0.2m.     

Purification and Characterization of Cold Adapted Trypsins from Antarctic krill (Euphausia superba)

Three trypsins (TRY-ES) were purified from Antarctic krill (Euphausia superba) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography, with relative molecular mass of 28.7, 28.8 and 29.2 kDa respectively. The TRY-ES was inhibited by specific trypsin inhibitors (benzamidine, STI, CHOM and TLCK), with optimum temperature at 40 (Trypsin I), 45 (Trypsin II) and 40 °C (Trypsin III) repetitively. The TRY-ES was stabled between 5 and 40 °C, which was consistent with the red shift in fluorescence intensity peak at 40 °C (Trypsin I) and 45 °C (Trypsin II and Trypsin III) and blue shift at 40 °C (Trypsin II and Trypsin III). The K _cat/K _m values of the TRY-ES was 14.28, 9.46 and 5.93 mM^?1s^?1 respectively, 1.1–10.2 folds higher than trypsins from other crustacean and mammal, which was supported by the differences in thermodynamics parameters, the free energy, enthalpy, and entropy of benzamidine and the TRY-ES system.     

Purification and characterization of β-Mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity

Marine bacterium Reinekea sp. KIT-YO10 was isolated from the seashore of Kanazawa Port in Japan as a seaweed-degrading bacterium. Homology between KIT-YO10 16S rDNA and the 16S rDNA of Reinekea blandensis and Reinekea marinisedimentorum was 96.4 and 95.4%, respectively. Endo-1,4-β-D-mannanase (β-mannanase, EC 3.2.1.78) from Reinekea sp. KIT-YO10 was purified 29.4-fold to a 21% yield using anion exchange chromatography. The purified enzyme had a molecular mass of 44.3?kDa, as estimated by SDS-PAGE. Furthermore, the purified enzyme displayed high specificity for konjac glucomannan, with no secondary agarase and arginase activity detected. Hydrolysis of konjac glucomannan and locust bean gum yielded oligosaccharides, compatible with an endo mode of substrate depolymerization. The purified enzyme possessed transglycosylation activity when mannooligosaccharides (mannotriose or mannotetraose) were used as substrates. Optimal pH and temperature were determined to be 8.0 and 70?°C, respectively. It showed thermostability at temperatures from 20 to 50?°C and alkaline stability up to pH 10.0. The current enzyme was thermostable and thermophile compared to the β-mannanase of other marine bacteria.     

Biodegradation of feather wastes and the purification and characterization of a concomitant keratinase from Paecilomyces lilacinus

Paecilomyces lilacinus strain PL-HN-16 was found to have the ability to degrade feathers. During the degradation process, the broth initially turned as sticky as gelatin and then turned into fluid that means the feathers can be hydrolyzed completely. Keratinolytic protein (Ker) of aforementioned strain was purified using ammonium sulphate precipitation, HiTrap? Butyl FF chromatography and Sephacryl S-200 gel filtration. The Ker of P. lilacinus PL-HN-16 had molecular mass of 33 kDa, the optimum pH 8.0 and temperature optimum at 40°C. It used the soluble keratin as substrate. The enzyme showed high activity and stability over a wide range of pH (6.0 to 10.0) and temperature (30°C to 60°C) values but was completely inhibited by PMSF. Ker of P. lilacinus PL-HN-16 exhibited stability toward SDS. These promising properties make the enzyme a potential candidate for future applications in biotechnological processes as keratin hydrolysis and dehairing during leather processing.     

Another Pectate Lyase Produced by Streptomyces nitrosporeus

Another pectate lyase was purified to a nearly homogeneous state from the culture filtrate of Streptomyces nitrosporeus. The molecular weight was estimated to be about 41,000. Iso-electric point was pH 4.6. The enzyme was most active at pH 10.0 and 50°C, and was relatively stable at a pH range of 4–11 (at 2°C for 48 hr) and below 40°C (at pH 7.0 for 10 min). Ca²⁺ was required for maximum activity. The enzyme was an endo-pectate lyase which was more active on low methoxyl pectin than on polygalacturonic acid and had macerating activity on potato tissue and Ganpi bark.