Tricine-sodium Dodecyl Sulfate-polyacrylamide Gel Research Articles

The structural characteristics and physicochemical properties of mung bean protein hydrolysate of protamex induced by ultrasound.

The limited physicochemical properties (such as low foaming and emulsifying capacity) of mung bean protein hydrolysate restrict its application in the food industry. Ultrasound treatment could change the structures of protein hydrolysate to accordingly affect its physicochemical properties. The aim of this study was to investigate the effects of ultrasound treatment on the structural and physicochemical properties of mung bean protein hydrolysate of protamex (MBHP). The structural characteristics of MBHP were evaluated using tricine sodium dodecylsulfate-polyacrylamide gel electrophoresis, laser scattering, fluorescence spectrometry, etc. Solubility, fat absorption capacity and foaming, emulsifying and thermal properties were determined to characterize the physicochemical properties of MBHP. MBHP and ultrasonicated-MBHPs (UT-MBHPs) all contained five main bands of 25.8, 12.1, 5.6, 4.8 and 3.9 kDa, illustrating that ultrasound did not change the subunits of MBHP. Ultrasound treatment increased the contents of α-helix, β-sheet and random coil and enhanced the intrinsic fluorescence intensity of MBHP, but decreased the content of β-turn, which demonstrated that ultrasound modified the secondary and tertiary structures of MBHP. UT-MBHPs exhibited higher solubility, foaming capacity and emulsifying properties than MBHP, among which MBHP-330 W had the highest solubility (97.32%), foaming capacity (200%), emulsification activity index (306.96 m2 g-1 ) and emulsion stability index (94.80%) at pH 9.0. Ultrasound treatment enhanced the physicochemical properties of MBHP, which could broaden its application as a vital ingredient in the food industry. © 2023 Society of Chemical Industry.

Design, expression and characterization of lactiscin—A novel broad-spectrum peptidic bacteriocin

Bacteria-derived antimicrobial peptides known as peptidic bacteriocins offer a promising alternative to traditional antibiotics in the face of the emergence of multidrug-resistant bacteria. Here, a nucleotide sequence of the gene encoding Lactococcus lactis-derived peptidic bacteriocin designated as lactiscin selectively identified from the GenBank® database was synthesized with an added 6⋅His sequence and cloned into Escherichia coli. Upon low-temperature expression at 16 °C, the His-tagged peptide could be produced in both soluble form and insoluble inclusions. Efficient purification of the soluble His-tagged peptide was achieved via immobilized-Ni2+ affinity chromatography (IMAC) and its estimated molecular mass of ∼13.4 kDa was determined by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified peptide was highly active against both Gram-positive and Gram-negative bacteria as it exhibited a minimal inhibitory concentration of 0.45 mg/mL, 0.15 mg/mL, 0.35 mg/mL and 0.45 mg/mL against. Escherichia coli, Vibrio parachemolyticus, Staphylococcus aureus and Micrococcus luteus, respectively. In addition, the lactiscin peptide still retained antimicrobial activity over a pH range of 3.0–12.0 and heat stability of 100 °C for 30 min. A membrane integrity study revealed that this peptidic bacteriocin was able to induce E. coli membrane permeabilization in a concentration-dependent manner, albeit it showed a negligible toxic effect on erythrocytic cells. Gel retardation assay demonstrated that the lactiscin bacteriocin could suppress the migration of genomic DNA extracted from pathogenic bacteria, suggesting the presence of bacteriocin-responsive binding genomic. Our findings of lactiscin—a novel broad-spectrum bacteriocin would be a valuable additive for the application of food industry as a potential bio-preservative.

Isolation, characterization, bacteriocin production and biological potential of Bifidobacteria of ruminants

Bacteriocins exhibited a wide spectrum of antibacterial activity against different pathogens. The aim of current study was to characterize the bacteriocins produced by Bifidobacterium spp. isolated from ruminants. The Bifidobacterium isolates were identified as B. longum, B. pseudolongum, B. bifidum, B. thermophilum, B. boum, B. merycicum and B. ruminantium. Bacteriocins were found to be pH stable, heat resistant, highly diffusible, NaCl tolerant and resistant to UV radiations. SDS, EDTA and urea induced 14%, 21% and 24% bacteriocins activity loss. Modified MRS broth (1% tryptone, 1% yeast extract and 2% glucose) was found to be the best nutrient medium for optimal production of bacteriocins. Minimum inhibitory concentration (MIC) values varied from 300 μl/ml to 500 μl/ml and minimum bactericidal concentration (MBC) values ranged from 500 μl/ml to >500 μl/ml for E. coli and S. aureus respectively. The highest protein concentration (29.0248 mg/ml) was recorded for Bifidobacteria bacteriocin produced by B. longum. Tricine-Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) revealed that molecular weight of isolated bifidobacterial bacteriocins was in the range of 3.6 kDa–30 kDa. Current study indicated that bifidobacterial bacteriocins have considerable potential to be used as biopreservative.

Purification and Partial Characterization of Bacteriocin Lac-B23, a Novel Bacteriocin Production by Lactobacillus plantarum J23, Isolated From Chinese Traditional Fermented Milk.

The exploration and evaluation of bacteriocin-producing lactic acid bacteria (LAB) have been one of the powerful means to food preservation. A total of 300 strains were isolated from Chinese traditional fermented milk products. A bacteriocin-producing LAB, named Lactobacillus plantarum J23, was screened and identified. Bacteriocin Lac-B23 from L. plantarum J23 was purified by 80% ammonium sulfate precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography. Molecular weight of bacteriocin Lac-B23 was determined to be approximately 6.73 kDa by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and it was confirmed as a novel bacteriocin by liquid chromatography-mass spectrometry. Moreover, bacteriocin Lac-B23 showed thermal stability when heated at below 100°C for 30 min, pH stability between pH 2.0 and 12.0, and sensitivity to trypsin, proteinase K, and proteinase E. The antimicrobial activity of bacteriocin could be enhanced by addition of Fe2+, Mn2+, and ethyl alcohol, and inhibited by Cu2+, K+, Ca2+, Zn2+, Mg2+, and sodium chloride. The results suggested bacteriocin Lac-B23 to have potential application prospects in the food industry.

Acid whey proteolysis to produce angiotensin-I converting enzyme inhibitory hydrolyzate

α-Lactalbumin (ALA) and β-lactoglobulin (BLG) are the main proteins in acid whey and proteolytic digestion of these proteins provides a source of peptides that inhibit angiotensin-I converting enzyme (ACE-I). Enzymatic hydrolysis of acid whey proteins using cysteine peptidase enzyme extract (antiacanthain) from ripe Bromelia antiacantha Bertol fruit was investigated. The effect of pH on hydrolysis kinetics of ALA and BLG by antiacanthain was analysed at 50 °C. Antiacanthain had a high catalytic efficiency for ALA (1.3 × 104 M−1 s−1) and BLG (1.7 × 104 M−1 s−1) at pH 9.2. The effect of temperature and amount of antiancanthain (enzyme to substrate ratio or E/S) on acid whey hydrolysis were studied by measurement of degree of hydrolysis (DH) and protein degradation profile evaluated by tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine–SDS-PAGE). Optimal acid whey hydrolysis was at pH 9.2, E/S = 1.0 (U/mg) and 50 °C. BLG was degraded faster than that of ALA, but after 30 min hydrolysis almost all BLG and ALA were degraded and new bands corresponding to peptides with a mass lower than 3.0 kDa appeared. Fractions of acid whey (DH = 12.3%), ALA (DH = 17.3%) and BLG (DH = 30.6%) hydrolyzates showed ACE-I inhibitory activity. Results also suggest that most inhibitory activity might be due to low molecular weight peptides from ALA, since 3 kDa permeate of ALA hydrolyzate showed the highest IC50 value (1.7 ± 0.4 µg/mL). Interestingly, low molecular weight peptides from both ALA and BLG had low immunoreactivity levels, monitored by latex agglutination tests, suggesting that these peptides could be safe for use by those with strong allergic responses. Results indicate that antiacanthain can be used as an enzymatic preparation for acid whey hydrolysis. The potential use of this hydrolyzate for manufacture of health-promoting products of high added value is discussed.

Isolation and Characterization of Lactoferrin Peptides with Stimulatory Effect on Osteoblast Proliferation

Lactoferrin is reported to be a potential food protein with osteogenic activity. However, the activity of lactoferrin peptides is questionable. In the present study, we isolated and characterized peptides from lactoferrin with stimulatory effect on osteoblast proliferation. Peptides from the lactoferrin pepsin hydrolysate were purified using cation-exchange and gel-filtration chromatography. Effects of different hydrolysates and peptides on the proliferation of osteoblast MC3T3-E1 cells were compared by MTT assay. Results showed that fraction P5-a from Superdex Peptide 10/300 GL gel chromatography showed better activity. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry confirmed that two peptides components of P5-a corresponded to fractions of 20-78 and 191-277 amino acids in Bos taurus lactoferrin molecule (GI: 221706349). These results will provide some theoretical and practical data for the preparation and application of osteogenic peptides in functional food industry.

Electrophoretic Characterization of Venom Proteins of <em>Montivipera xanthina</em> (Gray, 1849) (Ophidia: Viperidae)

In this study, with the aim of evaluating coagulant activities in the venom of M. xanthina, we analyzed venom proteins, digestion patterns of fibrinogen chains with venom and the effects of protease inhibitors on M. xanthina venom proteases using Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis. Venom samples were obtained from four adult specimens of Montivipera xanthina collected in Gümüldür (Izmir, Turkey). SDS-PAGE analysis demonstrated that 17 protein bands in the range of 20–250 kDa were present. The specific digestion patterns of fibrinogen chains revealed that M. xanthina venom possesses fibrinogenolytic enzymes which could be included in coagulation processes during envenomation Fibrinogenolytic activity directed exclusively towards the Aa-chain with a time-dependent activity towards Bb-chains suggests the presence of both metalloproteinases and serine proteases in M. xanthine venom. In the present study, the occurrence and inhibition of fibrinogenolytic activity of M. xanthina venom were clearly observed. For further analysis, the isolation, identification, and characterization of individual venom components will provide insight into their function and biological roles.

Biocontrol Activity of Bacillus subtilis Isolated from Agaricus bisporus Mushroom Compost Against Pathogenic Fungi.

Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom compost infected by red bread mold, exhibited antagonistic activities against Neurospora sitophila. Antifungal activity against phytopathogenic fungi was also observed. The maximum antifungal activity was reached during the stationary phase. This antifungal activity was stable over a wide pH and temperature range and was not affected by proteases. Assay of antifungal activity in vitro indicated that a purified antifungal substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In addition, treatment with strain B154 in A. bisporus mushroom compost infected with N. sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-associated laser desorption ionization time-of-flight mass spectrometry, and electrospray ionization tandem mass spectrometry analyses revealed a molecular weight consistent with 1498.7633 Da. The antifungal compound might belong to a new type of lipopeptide fengycin.

Conjugation of a peptide to mannan and its confirmation by tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The conjugation of polysaccharides to peptides is essential for antigen delivery and vaccine development. Herein, we show that tricine SDS-PAGE in combination with Coomassie Blue staining was adequate to determine the conjugation efficacy of a peptide (epitope 35–55 of myelin oligodendrocyte glycoprotein) to mannan. In addition, tricine SDS-PAGE and periodic acid–Schiff stains were able to monitor the redox state of mannan. Using the described protocol, more than 99.9% of a peptide containing five lysines at its N-terminus was confirmed conjugated to mannan.

Isolation and Characterization of Antithrombin Peptides from the Saliva of Malaysian Leeches

Purpose: To isolate and characterize the antithrombin compounds of Malaysian leeches’ saliva collection (LSC) for use as anticoagulant proteins and peptides.Method: Reversed phase - high performance liquid chromatography (RP-HPLC) was used to isolate all proteins from LSC. All isolated proteins were tested for anticoagulant activity by applying two tests, thrombin time test and the inhibition of the amidolytic activity of thrombin on the chromogenic substrate (S-2238). The molecular weights of isolated active compounds were identified using tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE).Results: More than 40 proteins were isolated from LSC using RP-HPLC. Two compounds (protein 1 and protein 2) were found to be active as they increased thrombin time by 30.26 and 36.75%, respectively and inhibited the amidolytic activity of thrombin. as evaluated by measuring the conversion of the chromogenic substrate (S-2238). Decrease in the conversion of the substrate (S-2238) by 31.10 and 41.61%, respectively was observed. The molecular weights of the proteins (6.289 and 14.255 kDa, respectively) had no similarity with other leech species.Conclusion: Two antithrombin proteins with anticoagulant properties were isolated from the saliva of Malaysian leeches using one-step isolation procedure. The proteins did not show any similarity to previous isolated proteins from leech.Keywords: Leech, Saliva, Antithrombin, Anticoagulant, Peptides, Molecular weight

Characterization of the Bacteriocin J4 Produced by Bacillus amyloliquefaciens J4 Isolated from Korean Traditional Fermented Soybean Paste

In order to isolate the antibacterial bacteriocin-producing bacteria against Listeria monocytogenes, one of noxious and prevalent food-poisoning bacterium, 117 strains of indigenous ferment bacteria were isolated from Korean traditional fermented soybean paste (Doenjang) in Korea. Among the isolated strains, 4 isolates inhibited the growth of L. Monocytogenes. Strain J4, showing the strongest inhibition activity against L. Monocytogenes, was identified as a Bacillus amyloliquefaciens by 16S rDNA sequencing. The strain had a broad spectrum of antibacterial activity against various food-borne pathogens such as Micrococcus luteus, Vibrio parahaemolyticus, and Staphylococcus aureus besides L. monocytogenes. The bacteriocin J4 was purified by 60% ammonium sulfate precipitation, carboxymethyl (CM)-Sephadex column chromatography, and Sephadex G-100 gel filtration. The purified bacteriocin J4 was estimated to be 39 kDa molecular weight by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bacterocin J4 had antimicrobial activity in a wide pH range (2.0-12.0) and was heat-stable at 80oC for 20 min, but the activity of bacterocin was inactivated by heating at 100oC for 15 min. The antimicrobial activity of the bacteriocin J4 was completely abolished by treatment with proteolytic enzymes such as protease (type I and IX), papain, trypsin, proteinase K, and pepsin, but not when treated with α-amylase, α-glucosidase, and β-glucosidase. The encoding gene of the bacteriocin J4 was included in chromosomal DNA, not plasmid. The bacteriocin J4 had a bacteriolytic mechanism, resulting in cell wall degradation of L. Monocytogenes.

Purification and Characterization of Enterocin LR/6, a Bacteriocin from Enterococcus faecium LR/6

Enterocin LR/6, a bacteriocin obtained from the culture filtrate of Enterococcus faecium strain LR/6, has been purified to homogeneity using ammonium sulfate precipitation, cation-exchange chromatography, gel-filtration, and checked on reverse-phase high-performance liquid chromatography. It is active at high temperatures (boiling as well as autoclaving) and over a wide range of pH (2.0-8.0). Also, it is sensitive to a number of proteolytic enzymes but is stable in the presence of surfactants and organic solvents. The protein could be stored at least up to 1 year at low temperatures (4 degrees C and -20 degrees C) without any loss of activity. The N-terminal sequence of enterocin LR/6 showed no homology with known enterocins or other bacteriocins present in the database, suggesting it to be a novel enterocin. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed its mass to be approximately 6.1 kDa. It showed a bactericidal mode of action against indicator strain, Micrococcus luteus.

Induction, selection and antibacterial activity of the antibacterial peptides from lepidopteran insect cultured cell lines

We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coli DH5α to stimulate the antibacterial peptide followed by antibacterial activity assay, induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment. The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest, and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation. A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 cells in 16 hours after stimulation. Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus, Escherichia coli K12D31 and Salmonella derby. It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli K12D31 and Salmonella derby.

Purification of bacteriocin LS1 produced by human oral isolate Lactobacillus salivarius BGHO1

Lactobacillus salivarius BGHO1, a human oral isolate with antagonistic activity against growth of Streptococcus mutans, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, and Salmonella enteritidis, probably produces more than one proteinaceous antimicrobial substance. The objective of this study was the purification of a bacteriocin, named LS1, produced by L. salivarius BGHO1. A simple and fast procedure for bacteriocin purification was developed, consisting of reverse-phase chromatography of the ammonium sulfate precipitate of cell-free culture supernatant by fast protein liquid chromatography and high-performance liquid chromatography, followed by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with the subsequent extraction of bacteriocin from the gel. The supernatant of L. salivarius BGHO1 culture retained its antimicrobial activity after boiling in a water bath for 15 min. Its antimicrobial activity was also maintained even after treatment for 20 min at 121 degrees C in an autoclave. Bacteriocin LS1 was purified to homogeneity. The molecular mass of bacteriocin LS1 was estimated to be approximately 10 kDa, based on tricine SDS-PAGE. During purification, another compound with antimicrobial activity, produced by L. salivarius BGHO1, was detected. The molecular mass of this compound was estimated to be approximately 5 kDa, based on tricine SDS-PAGE. Our results imply that LS1 is most probably a new bacteriocin, different from previously described bacteriocins produced by L. salivarius strains. The purification of bacteriocin LS1 enabled the further characterization of LS1 on both the molecular and genetic levels.

Improved adaptation to heat, cold, and solvent tolerance in Lactobacillus plantarum

The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40 degrees C) and cold shock (12 degrees C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.

Egg Yolk Protein and Egg Yolk Phosvitin Inhibit Calcium, Magnesium, and Iron Absorptions in Rats

Egg yolk decreases the absorption of iron. The effects of egg yolk protein and egg yolk phosvitin on the absorption of calcium, magnesium, and iron were investigated by in vivo studies. Male Wistar rats were fed purified diets containing casein, soy protein, or egg yolk protein for 14 d. The apparent absorptions of calcium, magnesium, and iron in the rats fed the yolk protein-based diet were lower than those in rats fed the casein- and soy protein-based diets. The apparent phosphorus absorption and the apparent protein digestibility in the yolk protein group were lower than those in the casein and soy protein groups. In the feces of the yolk protein group, serine comprised more than 30% of the amino acids. The addition of egg yolk phosvitin to the casein diets at levels of 1% and 2% (w/w) produced effects on calcium and magnesium absorptions similar to those produced by the diet containing yolk protein. The tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern suggested that phosphopeptide fragments having molecular masses of 28, 22, and 15 kDa were evident in the contents of the small intestine of the rats fed phosvitin diets. These results indicate that yolk protein, when compared with casein and soy protein, decreases calcium and magnesium absorption via the resistance of phosvitin to proteolytic action.

Separation of iron-binding protein from whey through enzymatic hydrolysis

Whey protein concentrate (WPC) was hydrolyzed by nine proteolytic enzymes to examine the effectiveness of the hydrolysates to bind iron. Degree of WPC hydrolysis was higher with pancreatin (13.91%), alcalase (13.60%), and flavourzyme (12.80%) compared with other enzymes (esperase, neutrase, papain, pepsin, protease and trypsin). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography analyses revealed maximum hydrolysis of α-LA and β-LG with alcalase. Molecular masses of peptides derived from alcalase hydrolysate were smaller than 6.5 kDa. Iron-binding by alcalase hydrolysate was the highest (97.6%) of all other hydrolysates. Using ion-exchange chromatography alcalase hydrolysate was eluted at a 0.25 m NaCl gradient concentration with higher iron-binding ability. This eluted fraction had higher Lys (18.09%), Ala (17.24%), and Phe (16.58%) contents. Alcalase showed noticeably better effectiveness than other enzymes to produce a hydrolysate for the separation of iron-binding peptides derived from WPC.

Molecular Characterization of Peptides Released from β-Lactoglobulin and α-Lactalbumin via Cardosins A and B

Crude mixtures of aspartic proteases from flowers of the plant Cynara cardunculus have been studied frequently, as have activities of such enzymes (in pure form) on caseins from bovine, ovine, and caprine sources. This research study addressed pure bovine whey protein as substrates; that is, α-lactalbumin (αLA) and β-lactoglobulin (α-LG), submitted to hydrolysis by 1 of 2 aspartic proteases (cardosins A and B), previously extracted and purified from C. cardunculus. Samples collected, following incubation at 55°C and pH 5.2, were assayed by fast protein liquid chromatography, reversed phase-high performance liquid chromatography, and tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis; the major peptides released were then collected and sequenced by Edman degradation. Cardosin B and, to a lesser degree, cardosin A showed proteolytic activity toward α-LA, but the hydrolyzates produced were characterized by distinct peptide profiles. Cardosin B possesses a broad specificity, and produces several hydrophobic peptides (at least 5, with molecular mass in the range 2 to 8 kDa) in the early stages, which eventually become more hydrophilic (with molecular mass below 2 kDa) at later stages of hydrolysis. Cardosin A was found to cleave α-LA at the peptide bonds Phe28-Arg29, Gly54-Tyr55, Ala59-Ile60, Leu71-Phe72, and Leu105-Thr106, whereas cardosin B cleaved Ala19-Glu20, Phe28-Arg29, Glu30-Leu31, Tyr37-Gly38, Trp45-Val46, Phe50-His51, Ala59-Ile60, Ser66-Thr67, Leu71-Phe72, Phe72-Gln73, Gln73-Ile74, Ile78-Trp79, Leu115-Asp116, and Leu124-Ala125. Conversely, cardosins A and B are apparently not active on β-LG.

Durancin L28-1A, a new bacteriocin from Enterococcus durans L28-1, isolated from soil

To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil. Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant. Enterococcus durans L28-1 produced a new bacteriocin. This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.

Serine/threonine phosphorylation associated with hamster sperm hyperactivation.

Background and Aims: Mammalian sperm activation and hyperactivation is regulated by protein phosphorylation. Although tyrosine phosphorylation is considered very important, several studies have investigated whether serine and threonine phosphorylation are also associated with sperm activation and hyperactivation, and that was also the aim of the present study. Methods: Protein phosphorylation of hamster spermatozoa was detected by Western blotting using antiphospho-amino acid monoclonal antibodies after tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences were analyzed using a peptide sequencer. Results: Four proteins were phosphorylated at serine residues during hyperactivation via activation and their approximate molecular weights were 90, 38, 32 and 10kDa, respectively. Five proteins were phosphorylated or dephosphorylated at threonine residues and their approximate molecular weights were 90, 70, 65, 35 and 10kDa, respectively. The 10-kDa protein corresponded to a previously reported 10-kDa tyrosine phosphoprotein. N-terminal sequences of the 10-kDa protein were similar to carcinustatin, which is a neuropeptide. Conclusions: During hyperactivation, four serine phosphorylation and five threonine phospho- or dephosphorylations occurred, which suggested that the 10-kDa protein was phosphorylated at tyrosine residues when spermatozoa were activated and then dual-phosphorylated at the serine and threonine residues during hyperactivation. (Reprod Med Biol 2004; 3: 223-230).