Purification and identification of a protease inhibitor from glassfish (Liparis tanakai) eggs

Two protease inhibitors of 67 and 18 kDa, respectively, were purified from glassfish, Liparis tanakai, eggs by affinity chromatography. The smaller protein was purified with a yield and purity of 0.25% and 49.69-fold, respectively, and was characterized for further study. The glassfish egg protease inhibitor exhibited stability between 50 and 65 degrees C in an alkaline environment (pH 8). It was shown to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 4.44 nM. Potent glassfish protease inhibitor with N-Val-Gly Ser-Met-Thr-Gly-Gly-Phe-Thr-Asp-C amino acid residues was synthesized and its inhibitory activity was compared. Moreover, the 18-kDa protein inhibited cathepsin, a cysteine protease, more effectively than did egg white protease inhibitor, whereas the reverse was true for papain. Glassfish egg protease inhibitor is classified as a member of the family I cystatins.     

Purification and partial characterization of a second cysteine proteinase inhibitor from ungerminated barley (Hordeum vulgare L.)

It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer.     

Partial isolation and characterization of a cysteine proteinase inhibitor from Lima bean (Phaseolus lunatus)

Lima beans (Phaseolus lunatus) have been shown to contain cysteine proteinase inhibitor (CPI) activity, but the CPI has not been isolated or characterized. Accordingly, our objective was to isolate and partially characterize a CPI from lima bean. The isolation scheme included water extraction of lima bean flour followed by a chromatography series using DEAE Sepharose, Phenyl Sepharose, hydroxyapatite, and reversed-phase high performance liquid chromatography. This scheme resulted in the partial purification of a approximately 20 000-dalton protein with high inhibitory activity against papain. This isolated lima bean CPI had an N-terminal sequence homologous with other members of the cystatin class of CPIs. The protein was relatively heat labile; suggesting it could be inactivated with normal cooking, which is favorable for its use in transforming plants to create insect resistance.     

Structure and stability of the potato cysteine protease inhibitor group (cv. Elkana)

The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.     

Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

Purification and characterization of a trypsin inhibitor from Plathymenia foliolosa seeds

A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDSPAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.     

Acute effects of wood-pulp on sockeye salmon (Oncorhynchus Nerka)

Freshwater suspensions of pulp fiber in concentrations exceeding 1000 ppm at 15°C are acutely lethal to sockeye salmon fingerlings. Limited adaptation of such fish to fiber is possible but injury, at least in the short term, is irreversible. The acute lethality of various toxicants associated with industrial pulps is synergistically enhanced by the presence of fiber, and pulp storage up to 158 days at 15°C had no significant effect on the acute lethality. Highly purified fibers are less toxic in fresh than in sea water. The sensitivity to fiber of fish from two sources did not differ significantly.     

The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family

The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.     

Structural characterization of potato protease inhibitor I (Cv. Bintje) after expression in Pichia pastoris

In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating. A cDNA coding for PI-1 from cv. Bintje was cloned and expressed in Pichia pastoris. Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature. The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins. Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit. The transition temperature for the recombinant PI-1 was 88 degrees C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje.     

Purification and characterization of amylases from small abalone (Sulculus diversicolor aquatilis)

Amylases II-1 and II-2 with molecular weights of 55.7 and 65 kDa, respectively, were purified to electrophoretical homogeneity from small abalone (Sulculus diversicolor aquatilis) by ammonium sulfate fractionation, Sepharose CL-6B, CM-Sepharose CL-6B, and Sephacryl S-100 chromatographs. They had optimal temperatures of 45 and 50 degrees C and an optimal pH of 6.0. The purified amylases were stable at pH 5.0-8.0 and 6.0-8.0, respectively. They were completely or partially inhibited by Hg(2+), Cu(2+), Cd(2+), Zn(2+), iodoacetamide, phenylmethanesulfonyl fluoride, and N-ethylmaleimide, suggesting the existence of cysteine at their active sites. Digestion tests against various polysaccharides suggested that the purified amylases II-1 and II-2 are neoamylases which can hydrolyze both alpha-1,4 and alpha-1,6 glucosidic bonds. Amylase II-2 might be an exo- and II-1 an endo-/exo-amylase.     

Alaska pink salmon (Oncorhynchus gorbuscha) spoilage and ethanol incidence in the canned product

Ethanol was quantified in canned salmon produced from whole fish showing different stages of decomposition due to storage at 1 and 14 degrees C for up to 3 and 16 days, respectively. Ethanol incidence in the canned salmon was correlated to results from skin aerobic plate counts and sensory evaluations of the whole fish and with sensory evaluations of the canned product. Panelists rejected whole salmon after 3 and 12 days of storage at 14 and 1 degrees C, respectively. Skin aerobic plate counts reached 4.8 log CFU/cm2 when fish were rejected, regardless of storage temperature. Panelists rejected canned salmon produced with fish stored for a maximum of 2 and 16 days at 14 and 1 degrees C, respectively. Ethanol concentrations in the cans produced with fish stored at 14 degrees C correlated well with sensory evaluation results; however, ethanol concentrations in the cans produced with salmon stored at 1 degrees C did not agree with sensory results. A correlation could not be established between ethanol concentration in the canned product and microbial content of whole salmon.     

Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas)

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.     

Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon)

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

Kunitz-type serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung)

An antifungal protein, AFP-J, was purified from tubers of the potato (Solanum tuberosum cv. L Jopung) by various chromatographic columns. AFP-J strongly inhibited yeast fungal strains, including Candida albicans, Trichosporon beigelii, and Saccharomyces cerevisiae, whereas it exhibited no activity against crop fungal pathogens. Automated Edman degradation determined the partial N-terminal sequence of AFP-J to be NH2-Leu-Pro-Ser-Asp-Ala-Thr-Leu-Val-Leu-Asp-Gln-Thr-Gly-Lys-G lu-Leu-Asp-Ala-Arg-Leu-. The partially sequence had 83% homology with a serine protease inhibitor belonging to the Kunitz family, and the protein inhibited chymotrypsin, pepsin, and trypsin. Mass spectrometry showed that its molecular mass was 13 500.5 Da. This protease inhibitor suppressed over 50% the proteolytic activity at 400 microg/mL. These results suggest that AFP-J is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.     

Purification and partial characterization of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps

A lipoxygenase from Terfezia claveryi Chatin ascocarp, a mycorrhizal hypogeous fungus, is described for the first time. The higher proportion of PUFA in T. claveryi ascocarps makes lipid rancidity the main factor limiting its storage life. Thus, the studies on LOX from T. claveryi are important because this enzyme, among other roles, may be involved in an alteration of lipids leading to consumer rejection. The enzyme has been purified to apparent homogeneity by phase partitioning in the presence of Triton X-114, followed by two steps of cation-exchange chromatography. The purified T. claveryi LOX preparation consisted of a single major band with an apparent molecular mass of 66 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic activity exhibited a strong specificity toward linoleic and linolenic acids as substrates, while only 32% activity was observed using gamma-linolenic acid. The pH optimum of this enzyme was pH 7.0. When the enzyme reacted with linoleic acid, it produced a single peak, which comigrated with standard 13-hydroperoxy-octadecadienoic acid; 13-hydroperoxy-octadecatrienoic acid was produced during the reaction with linolenic acid.