Isolation and partial characterization of rennet-like proteases from Australian cardoon (Cynara cardunculus L.)

The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.     

Beta-glucosidase from Chalara paradoxa CH32: purification and properties

The hyphomycete Chalara paradoxa CH32 produced an extracellular beta-glucosidase during the trophophase. The enzyme was purified to homogeneity by ion-exchange and size-exclusion chromatography. The purified enzyme had an estimated molecular mass of 170 kDa by size-exclusion chromatography and 167 kDa by SDS-PAGE. The enzyme had maximum activity at pH 4.0-5.0 and 45 degrees C. The enzyme was inactivated at 60 degrees C. At room temperature, it was unstable at acidic pH, but it was stable to alkaline pH. The purified enzyme was inhibited markedly by Hg(2+) and Ag(2+) and also to some extent by the detergents SDS, Tween 80, and Triton X-100 at 0.1%. Enzyme activity increased by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified beta-glucosidase was active against cellobiose and p-nitrophenyl-beta-D-glucopyranoside but did not hydrolyze lactose, maltose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside, or xyloside derivatives of p-nitrophenol. The V(max) of the enzyme for p-NPG (K(m) = 0.52 mM) and cellobiose (K(m) = 0.58 mM) were 294 and 288.7 units/mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (K(i) = 11.02 mM). Release of reducing sugars from carboxymethylcellulose by a purified endoglucanase produced by the same organism increased markedly in the presence of beta-glucosidase.     

Biochemical and biophysical changes induced by fungicide sodium diethyl dithiocarbamate (SDD), in phytocystatin purified from Phaseolus mungo (Urd): a commonly used Indian legume

Phytocystatins are the plant thiol protease inhibitors involved in several reaction mechanisms of the plant system like regulation of proteolytic activity and storage of proteins. Biochemical and biophysical changes induced by fungicide SDD in phytocystatin purified from Phaseolus mungo have been investigated in terms of mass spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy, at pH 7.0, with varying fungicide concentrations (1-9 mM) and a time of incubation ranging from 2 to 8 h at 37 degrees C, with a fixed cystatin concentration (1.5 mM). Reactive oxygen species responsible for inhibitor damage were also investigated, and thiourea was found to scavenge the free radicals generated by SDD. FTIR analysis indicates a significant conformational transition from alpha-helix to beta-sheet structure; quenching of fluorescence is evident by fluorescence spectroscopy. The activity assay showed a decrease in inhibitory activity, as well as a fragmentation of the inhibitor was observed in electrophoresis. Results obtained implicate that exposure of phytocystatins to SDD involves physicochemical changes in cystatins leading to damage and a decrease in the activity of the inhibitor.     

A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.     

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.     

Peptides from Lactobacillus hydrolysates of bovine milk caseins inhibit prolyl-peptidases of human colon cells

Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from α(S1)-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-amino-dipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects.     

Digestion of peptidic residues in humic substances by an alkali-stable and humic-acid-tolerant proteolytic activity in the gut of soil-feeding termites

Previous feeding experiments have shown that soil-feeding termites (Termitidae: Termitinae) preferentially mineralize the peptidic component of synthetic humic acids, but nothing was known about the mechanism involved in digestion. Here, we studied the hydrolysis of humus-stabilized peptides in gut extracts of Cubitermes orthognathus by measuring the release of radiolabel from ¹⁴C-peptide-labeled synthetic humic acids. Gut extracts exhibited proteolytic activity over a wide pH range (from 4 to 12) with a maximum at about pH 8. The highest activity was located in the gut section containing the midgut and the extremely alkaline (up to pH 12) mixed segment. Chemical hydrolysis at in situ pH (up to pH 12) was negligible. Proteolytic activity in the hindgut fluid was generally relatively low, but alkaline proteases dominated in the anterior hindgut. When compared to other alkaline proteases, the proteolytic activity of gut extracts had a higher alkali-stability and tolerance to humic acids than subtilisin and an alkaline protease of Streptomyces griseus. Gut extracts also hydrolyzed the peptidic component of synthetic humic acids more efficiently than the commercial enzymes. Together with previous results, this study strongly supports the hypothesis that soil-feeding termites mobilize and digest the peptidic component of organic nitrogen in soil humic substances by a combination of proteolytic activities and extreme alkalinity in their intestinal tract.     

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.     

Purification and characterization of gelatinase-like proteinases from the dark muscle of common carp (Cyprinus carpio)

Gelatinolytic proteinases from common carp dark muscle were purified by 30-60% ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q, and affinity on gelatin-Sepharose. The molecular masses of these proteinases as estimated by SDS-PAGE were 75, 67, and 64 kDa under nonreducing conditions. The enzymes revealed high activity at a slightly alkaline pH range, and their activities were investigated using gelatin as substrate. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the gelatinolytic activity, whereas other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca (2+) is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases hydrolyze native type I collagen effectively even at 4 degrees C, strongly suggesting their involvement in the texture softening of fish muscle during the post-mortem stage.     

Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization

A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry.     

Purification, characterization, and solvent-induced thermal stabilization of ficin from Ficus carica

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 +/- 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.     

Production, purification, and properties of an endoglucanase produced by the hyphomycete Chalara (Syn. Thielaviopsis) paradoxa CH32

The hyphomycete Chalara (syn. Thielaviopsis) paradoxa produces endoglucanase activity during the late trophophase. The low molecular mass (35 kDa) endoglucanase purified from cultured broths works optimally at 37 degrees C and pH 5.0. The enzyme inactivates at pH below 3.0 and also at temperatures of 50 degrees C or higher, but it is stable at lower temperatures, including refrigeration temperature and freezing. The enzyme is inhibited by detergents, by EDTA, and by the divalent cations Hg(2+) and Ag(2+). It is also inhibited to some extent by 10 mM Zn(2+), Fe(2+), and Mg(2+), but it is stimulated by Mn(2+). Enzyme activity is not affected by reducing agents. In the presence of low concentrations of water miscible organic solvents (20%) endoglucanase activity is inhibited by 7% (for methanol) to 50% (for acetonitrile), and it is totally inhibited at higher solvent concentrations (50%). Enzyme activity is not affected by the water immiscible solvent ethyl acetate. Carboxymethylcellulose is the preferred substrate (K(m(app)) = 8.3 g/L; V(max(app)) = 1.1 microM/min). Hydrolysis of crystalline cellulosic substrates is very limited, but it is greatly enhanced by phosphoric acid swelling. The purified enzyme shows no activity toward disaccharides or aryl-glucosides. Its activity is inhibited by cellobiose.     

Purification and biochemical properties of dipeptidyl peptidase I from porcine skeletal muscle

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.     

Cathepsin L: a predominant heat-activated proteinase in arrowtooth flounder muscle

Characterization of the autolytic profile of arrowtooth flounder (ATF) muscle indicated the involvement of heat-activated proteinases active at both acidic and alkaline pH values. Further assay of fish extract exhibited the maximum activity at 60 degrees C against casein used as a substrate at both pH 5.5 and 8.0. The maximum activity shifted to lower temperatures by the addition of urea with two distinctive patterns: activity reduction at pH 5.5 and activity enhancement at pH 8.0. The highest inhibition by E-64 indicated the proteinase belongs to the cysteine proteinase class. At pH 5.5, the proteinase hydrolyzed Z-Phe-Arg-NMec and all types of protein substrates tested at higher rate than that at pH 8.0. Activity bands, observed on the activity-stained substrate gels, indicated similar proteinases are responsible for the proteolytic activity observed at both pH values. When proteins of fish extract were separated by HPLC-SEC, only one proteolytic peak was observed at the retention time of 26 min with an estimated molecular weight of 39800 Da. The results implied cathepsin L is a predominant proteinase responsible for autolysis of ATF muscle at elevated temperatures.     

Evaluation of extracts from Gevuina avellana hulls as antioxidants

The antioxidant activity of the extracts from Gevuina avellana hulls was evaluated and compared with that of BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole), using the beta-carotene bleaching assay, the accelerated oxidation of crude soybean oil, and the 2,2-diphenyl-beta-picrylhydrazyl (DPPH) radical scavenging method. Solvents of different polarity were used to obtain the extracts. Both the extraction yield and the antioxidant activity were strongly dependent on the solvent. The ethanol and diethyl ether soluble fractions were the most active with the beta-carotene assay. Ethanol and methanol extracts were the most active in hydrogen radical scavenging activity. Water and methanol inhibited more efficiently the oxidation of soybean oil at 70 and 80 degrees C, respectively. As a general trend, increased antioxidant activity was observed for increased extract concentration. Except the acetone extracts, all were stable after 6 months storage at 4 degrees C. The ethanol solubles from G. avellana hulls present antioxidant activity similar to that of synthetic antioxidants and to other reported residual agroindustrial materials.     

Pressurized fluid extraction of bioactive compounds from Phormidium species

In the search for new functional ingredients with potential use in the food industry, extracts of unknown species of microalgae, such as Phormidium species have been studied. Three solvents of different polarities (i.e., hexane, ethanol, and water) have been used to obtain pressurized liquid extracts with different compositions. Moreover, extractions were performed at four different extraction temperatures (50, 100, 150, and 200 degrees C) with 20 min as extraction time. Antioxidant activity of the extracts has been measured by the TEAC assay. In general, hexane and ethanol extracts showed a higher antioxidant capacity that was mainly attributed to carotenoid compounds, as the TEAC value trend seems to be similar to the carotenoid content of the extracts. On the other hand, the high antioxidant activity of the 200 degrees C water extracts is likely related to the presence of Maillard reaction compounds produced by thermal degradation of the sample. beta-Carotene, lutein, violaxanthin, and neoxanthin were identified in 150 degrees C ethanol extracts. Four different microbial species ( Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger) were used to screen the potential antimicrobial activity of the Phormidium sp. extracts. The most sensitive microorganism was the yeast, C. albicans, whereas the fungus, A. niger, was the most resistant. In general, no drastic differences were found for solvents and temperatures tested, showing a very diverse nature of the compounds responsible for the antimicrobial activity of these microalgae. In ethanol extracts, antimicrobial activity could be mainly attributed to the presence of terpenes (i.e., beta-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) in the samples. Toxicity studies carried out with the extracts evaluated in the present work showed a cellular toxicity lower than those of other cyanobacteria such as Spirulina plantensis.     

Purification and characterization of lipase in buckwheat seed

To obtain basic information about enzymatic deterioration of buckwheat flour, triacylglycerol lipase (LIP; EC 3.1.1.3) was purified from buckwheat seed. The LIP consisted of two isozymes, LIP I and LIP II, and they were purified with purification folds of 60 and 143 with final specific activities of 0.108 and 0.727 mumol of fatty acid released per minute per milligram of protein at 30 degrees C using triolein as a substrate. Molecular weights were estimated to be 150 (LIP I) and 28.4 kDa (LIP I) by gel filtration and 171 (LIP I) and 26.5 kDa (LIP II) by SDS-PAGE. Optimal pHs of LIP activities were 3.0 (LIP I) and 6.0 (Lip II) using triolein as a substrate. Both LIP I and II reacted in the acidic pH range. Optimal temperatures were 30 (LIP I) and 40 degrees C (LIP II), and both LIP I and II were stable below 30 degrees C when p-nitrophenyl-laurate was used as a substrate. However, they were inactivated above 60 degrees C. On the other hand, when triolein was used as a substrate, optimal temperatures were 30 degrees C for both LIP I and II, and they retained 40% of their activity after a 4 h incubation of enzymes at 70 degrees C. LIP I and II had higher activity against triolein than monoolein or tri/monopalmitin. Most of the LIP activity was distributed in the embryo.     

Purification and characterization of bacteriocin from Pediococcus pentosaceus ACCEL

The Pediococcus pentosaceus ACCEL bacteriocin was purified to electrophoretical homogeneity by cell adsorption-desorption and Superose 12 fast performance liquid chromatography (FPLC). The purified bacteriocin, with a molecular mass of 17.5 kDa and an N-terminal sequence of -KYYGNGVTXGKHSXXVDXG-, belongs to class IIa and is designated pediocin ACCEL. It was inactivated by various proteases and stable at pH 2.0-6.0 and <100 degrees C. More than 80% activity was left even after 15 min of heating at 121 degrees C and pH 2.0-4.0. Gram-positive food-borne pathogens were inhibited by this bacteriocin, but Gram-negative ones were not. According to the storage stability study, the purified pediocin was stable at pH <6.0 and low temperature. No significant change in bactericidal activity was observed after freeze-drying and subsequent 1-month storage at room temperature.