Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

一株曲霉的一种中温糖化酶的纯化及其部分酶学特性

本研究从广西花坪自然保护区采集的土壤中筛选获得了一株产糖化酶丝状真菌菌株57-45,通过形态学观察和真菌内转录间隔区(internal transcribed spacer,ITS)序列比对分析,将其初步鉴定为曲霉属(Aspergillus sp.)的一个种。纯化真菌57-45所产的一种胞外糖化酶经过硫酸铵分级沉淀、疏水层析和阴离子交换层析三步蛋白质纯化步骤,得到在SDS-PAGE上约60kD的单一蛋白质条带,薄层层析分析表明该纯化的蛋白质水解可溶性淀粉的产物只有葡萄糖,证明该纯化的蛋白质为糖化酶。纯化的糖化酶Km值为1.9mg/mL,Vmax为4148μmol/(min·mg),最适作用pH5.5,最适作用温度50℃,在同步糖化发酵中有应用的潜力。金属离子Fe3+、Zn2+、Cu2+对酶活有较强的抑制作用,EDTA对酶活有较强的促进作用。本文结果将为进一步研究曲霉糖化酶的酶学特性提供新的材料。     

Purification and Characterization of Enterotoxin Produced by Aeromonas sobria

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.     

Purification and Characterization of Lipid Bioflocculant Produced by Rhodococcus erythropolis

The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.     

Purification and Characterization of Anti-Listeria Compounds Produced by Geotrichum candidum

Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes. These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified: d-3-phenyllactic acid and d-3-indollactic acid.

Contamination by Listeria has become a problem over the past 20 years in many parts of the world. The ubiquitous nature of Listeria monocytogenes, its capacity to multiply at refrigeration temperatures, its thermal tolerance (11), and its resistance to relatively low pH (it can multiply at pH 5.3 and 4°C and at pH 4.39 and 30°C) (5), together with its tolerance of high salt concentrations (4, 18), make controlling this potentially pathogenic microorganism in food products difficult. This bacterium has been incriminated in several cases of food poisoning (2, 10, 19). At risk are the immunodepressed, the old, pregnant women, fetuses, and newborn babies. Several groups have worked on biological control. As a result, many bacteriocins, which inhibit the growth of L. monocytogenes, have been isolated, purified, and characterized (12, 13, 16, 18). We have worked with Geotrichum candidum, a yeast-like member of the natural milk flora that is used as a maturing agent for soft and hard cheeses. In an extensive study carried out in 1984 (7), the interactions between G. candidum and the microflora in cheeses were examined. G. candidum inhibited the growth of gram-negative bacteria, gram-positive bacteria, and fungi (6). We recently showed (3) that G. candidum inhibits the growth of L. monocytogenes on both solid and liquid media (a bacteriostatic effect). The inhibitors are stable over a wide pH range and can be heated to 120°C for 20 min. The present report describes the purification and characterization of compounds responsible for this antibacterial action.Microorganisms, culture conditions, and detection of inhibitory activity.The strain of G. candidum used came from the collection of the Caen University Food Microbiology Laboratory, Caen, France (UCMA G91) and was initially isolated from a cheese, Pont l’Evêque. One percent of a preculture (optical density at 620 nm [Milton Roy Spectronic 301; Bioblock Scientific, Illkirch, France] of 0.7 [10⁷ arthrospores or hyphae/ml]) of G. candidum was grown in a fermentor (20 liters; Biolafitte type PI) in 15 liters of Trypticase soy broth (30 g/liter; Biomerieux, Marcy l’Etoile, France) with yeast extract (6 g/liter; AES, Combourg, France) (TSBYE) buffered to pH 6.3 with 0.1 M citrate-0.2 M phosphate. The culture was stirred at 300 rpm for 64 h at 25°C under a pressure of 0.2 bar and was then filtered through a 1,000-Da cut-off membrane by tangential ultrafiltration (Sartorius, Palaiseau, France) under a pressure of 2 bars. The resulting ultrafiltrate was sterilized by passage through a capsule (Sartorius) containing 0.45-μm- and 0.2-μm-pore-size membranes.The inhibition of L. monocytogenes was checked at each purification step by the agar diffusion well assay (3). Antimicrobial activity was estimated by measuring the diameter of the inhibitory halo on two right-angle axes (average of two plates). The strain of L. monocytogenes (UCMA L205) (serovar 1/2a; Centre National de Référence des Listeria, Nantes, France) and lysovar 1652 (Institut Pasteur, Paris, France) came from the laboratory collection and was isolated from milk. The initial lyophilized ultrafiltrate (900 mg/ml) gave a halo diameter of 36 ± 0.7 mm in the inhibition assay.Purification.Samples of ultrafiltrate (20 μl) were spotted on thin-layer chromatography (TLC) plates (silica gel, 10 by 5 cm, 0.25 mm thick, 60 F₂₅₄; Merck, Darmstadt, Germany), with 20 μl of TSBYE for controls, and eluted by vertical chromatography with a butanol-acetic acid-water (40:10:20 [vol/vol/vol]) solvent system. The bands were examined under UV light (254 nm) or after treatment with Ehrlich’s reagent. Four well-separated bands were found (R_fs, 0.11 ± 0.04; 0.41 ± 0.04; 0.7 ± 0.03; and 0.86 ± 0.03), but only the band with an R_f of 0.7 ± 0.03 differed from that of control preparations (TSBYE not containing G. candidum). The microbiological bioautography test (1) confirmed the presence of the inhibitor in the band with an R_f of 0.7. Lyophilized ultrafiltrate was subjected to centrifugal partition chromatography (Sanki 1000 Engineering Ltd.; EverSeiko, Tokyo, Japan) in butanol-acetic acid-water (40:10:50 [vol/vol/vol]). Partitioning was carried out under the following conditions: ascending mode, 1,200 rpm; flow rate, 3 ml/min; pressure, 40 bars. An aliquot of material (3 g) previously equilibrated with the solvent system was injected into the separatus via a 12-ml injection loop. Fractions (10 ml each) were collected and evaporated to dryness in a SpeedVac (Jouan RC 1022, Saint Herblain, France). The dried extracts of certain fractions were taken up in 800 μl of water and brought to pH 5.6 with 0.2 M NaOH. A total of 10 mg of pooled fractions with an R_f close to 0.7 and showing L. monocytogenes inhibitory activity (36 ± 0.7 mm for a solution of 38 mg/ml) was taken up in 250 μl of methanol-water (50:50 [vol/vol]) and automatically deposited (Camag Linomat) on a 10- by 20-cm TLC plate (silica gel, 0.25 mm thick, 60 F₂₅₄; Merck). The plate was developed with butanol-acetic acid-water (40:10:20 [vol/vol/vol]) and examined under UV light. Bands with an R_f of 0.7 were scraped off and placed in methanol. The silica was washed several times and removed by centrifugation and filtration through a 0.2-μm-pore-size filter. An aliquot (20 μl) was spotted on a small silica TLC plate to confirm elution of the solute by the methanol solvent. The purity of the band with an R_f of 0.7 was confirmed by high-pressure liquid chromatography (HPLC) coupled with a photodiode array detector at 206 and 222 nm (solvent A: 0.1% formic acid in water; solvent B: CH₃CN-H₂O [95:5] plus 0.1% formic acid) on a C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic, Herrenberg, Germany) at the flow rate of 1 ml/min. Inhibitory activity was assessed as above. Preparative TLC indicated that the band with an R_f of 0.7 contained two components, one eluting at 4.5 min on HPLC (peak 1) and the other at 5.5 min (peak 2). The latter fraction gave an inhibitory halo of 36 ± 0.7 mm at a concentration of 20 mg/ml (a 45-fold purification over the ultrafiltrate). The material from preparative TLC (40 mg in 200 μl of methanol-water) was placed on a column of Sephadex LH20 (1 m by 1 cm; Pharmacia), and the column was eluted with methanol-water at 12 ml h⁻¹. Fractions (1 ml each) were collected and examined by HPLC to determine the material in each fraction. This final purification on Sephadex LH20 gave two peaks, with two-thirds of the eluate at peak 1 and one-third at peak 2 (Fig. ​(Fig.1).1). As the concentration for peak 2 was very low, only the inhibitory activity for peak 1 was assayed. A concentration of 20 mg/ml gave a halo diameter of 26 ± 0.7 mm. Open in a separate windowFIG. 1Reverse-phase liquid chromatography (HPLC) of inhibitory compounds of G. candidum after purification by centrifugal partition chromatography, preparative TLC, and Sephadex LH20 gel filtration. Column, C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic). Eluent: solvent A (0.1% formic acid in water), solvent B (CH₃CN-H₂O [95:5] plus 0.1% formic acid). Flow rate, 1 ml/min. (a) Product 1 (detection at 206 nm); (b) product 2 (detection at 222 nm).

Characterization.

The pooled fractions were run on HPLC with a Grom-Sil ODS2 column coupled to a mass detector (Sciex Api III, triple quadrupole; Thornhill, Canada). Product 1, analyzed by desorption and chemical ionization, gave a signal at an m/z of 184 for (M⁺ NH₄)⁺ on desorption and chemical ionization and thus had a mass of 166. Product 2 was analyzed by ion spray and gave a signal at an m/z of 297 (M + 4 Na)⁺ for a mass of 205. Spectra were determined in a Nicolet model 60 SXR FT-IR. Samples were dissolved in dimethyl sulfoxide, and the ¹H and ¹³C resonances were measured in a Brucker spectrometer at 200 and 400 Hz, respectively. The purified material was taken up in methanol, and the isomeric form of the substance(s) inhibiting L. monocytogenes was determined in a Perkin-Elmer model 341 polarimeter. Two inhibitors were identified (Fig. ​(Fig.2);2); product 1 was 2-hydroxy-3-phenylpropanoic acid (phenyllactic acid, mass 166), and product 2 was 2-hydroxy-3-indolpropanoic acid (indollactic acid, mass 205). The rotation of polarized light showed that the phenyllactic acid produced by G. candidum was the d form. The spectrum properties of the isolated compounds are identical to those of authentic commercial compounds (Sigma Chemical Co., St. Louis, Mo.). d-Phenyllactic acid can be purchased from Aldrich (product no. 37 690-6), and dl-indollactic acid is available from Sigma (catalog no. I2875). Inhibitory activity with commercial compounds showed that dl-phenyllactic acid (Sigma catalog no. P7251) was a stronger inhibitor of Listeria than dl-indollactic acid (34 and 26 ± 0.7 mm for 187 mM, respectively) and that the d form of phenyllactic acid was more active (38 mm for 120 mM) than the l form (Aldrich 11, 306-9, 30 mm for 120 mM). The samples were taken up in methanol-water (50:50 [vol/vol]) and brought to pH 5.6 (Table ​(Table1).1). Open in a separate windowFIG. 2Structure of two inhibitory compounds of G. candidum characterized by LC-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation.TABLE 1Anti-Listeria activity of phenyllactic acid and indollactic acid^a

Extracellular Laccase Produced by an Edible Basidiomycetous Mushroom,Grifola frondosa: Purification and Characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K _m values of Lac 1 toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.     

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Summary Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The K_m and V_max values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.     

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Purification and Characterization of a Low-Molecular-Weight Xylanase Produced by Acrophialophora nainiana

A low-molecular-weight xylanase activity (XynI) was isolated from the fungus Acrophialophora nainiana after growth in a solid medium containing wheat bran. XynI was purified to apparent homogeneity by ultrafiltration and gel filtration chromatography. The purified enzyme had a molecular weight value of approx. 17 kDa, as determined by SDS-PAGE. This enzyme was most active at 50°C and pH 6.0. At 50°C the half-life was 150 min. The apparent K _m value for birchwood xylan was much lower than the K _m value for oat spelt xylan. XynI was activated by L-cysteine, DTE, β-mercaptoethanol, and L-tryptophan. XynI did not show significant sequence homology with other xylanases. The analysis of hydrolysis products of xylans and wood pulps showed that XynI was able to release xylooligomers ranging from X2 to X3 and X2 to X6, respectively. The enzyme was not active against acetylated xylan. A small amount of xylose was released from deacetylated, birchwood, and oat spelt xylans. The results obtained with enzymatic treatment of Kraft pulp indicated a reduction in the amount of chlorine compounds required for the process and enhanced brightness gain. Received: 6 May 1998 / Accepted: 29 July 1998     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Partial Purification and Characterization of a Bacteriocin Produced by Propionibacterium thoenii

A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 was found to be active against closely related species and exhibited a broad spectrum of activity against other microorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, and stable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfate precipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1 was present as two different protein aggregates with apparent molecular weights of more than 150,000 and approximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein common to both with an apparent molecular weight of 10,000.     

Glycerol Dehydrogenase from Cellulomonas sp. NT3060: Purification and Characterization

NAD⁺-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn²⁺, Cu²⁺ and Cd²⁺. Oxidation of glyberol was activated by Na⁺ and reduction of dihydroxyacetone was activated by K⁺ at pH 7.5.     

Purification and Characterization of the Antioxidative Substance Produced by Aspergillus sojae K

An antioxidative substance produced by Aspergillus sojae K is absolutely soluble in water and strongly inhibits autoxidation of Na-ascorbate. The substance, produced extracellularly in the culture ftuid by the mold, was purified by ion exchange chromatography, gel filtration, and HPLC. The substance was purified 34-fold with an activity recovery of 38% from culture fluid. Purity of the substance was confirmed with a single peak through HPLC using an analytical column as well as a single spot on TLC. The purified substance consists of equimolar aspartic acid and glycine, indicating that the substance is a peptide. From mass spectral analysis the molecular weight was 710, but the precise sequence of the amino acids is not clear. The substance is stable at 70°C, but about 80% of the activity was lost at 80°C after 60 min. Besides, the substance is completely stable at pH 3–14. This substance efficiently suppressed the oxidation of fish oil.     

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.     

Purification and Characterization of a Protease Produced by Streptococcus Faecalis Var. Liquefaciens

Abstract

An extracellular protease produced by Streptococcus faecalis var. liquefaciens has been purified 535 fold by standard purification procedures. The homogeneity of the enzyme preparation was demonstrated by disc electrophoresis, isoelectrofocusing, sedimentation equilibrium and the Ouchterlony technique. The enzyme has a molecular weight of approximately 30,000 with an isoelectric point of 4.7. Kinetic values with casein as a substrate, show it to have a K of 9.1 × 10 ^?2mmoles with a V_max of 0.454 ΔO.D. max     

Purification and Characterization of a Cysteine Protease Produced by Pathogenic Luminous Vibrio harveyi

The purification and characterization of an extracellular protease produced by pathogenic luminous Vibrio harveyi strain 820514, originally isolated from diseased tiger prawn (Penaeus monodon), was presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of hydrophobic interaction chromatography and anion exchange on fast protein liquid chromatography. The protease is an alkaline cysteine protease, heat labile, inhibited by iodoacetamide, iodoacetic acid, N-ethylmaleimide, p-chloromercuribenzoate, and p-chloromercuribenzene-sulfonic acid, and showed maximal activities at pH 8 and 50°C, having a molecular mass of 38 kDa as estimated by SDS-PAGE and gel filtration column. In addition, the protease was also completely inhibited by CuCl₂ and HgCl₂, but not or only partially inhibited by other inhibitors tested. Furthermore, 2-mercaptoethanol was the most effective reducing agent in the activation of the enzyme. The present protease is the first cysteine protease found in Vibrio species. Received: 20 November 1996 / Accepted: 7 January 1997     

Purification and Characterization of Bacteriocin Produced by Weissella confusa A3 of Dairy Origin

A dramatic increase in bacterial resistance towards currently available antibiotics has raised worldwide concerns for public health. Therefore, antimicrobial peptides (AMPs) have emerged as a promisingly new group of therapeutic agents for managing infectious diseases. The present investigation focusses on the isolation and purification of a novel bacteriocin from an indigenous sample of cow milk and it’s mode of action. The bacteriocin was isolated from Weissella confusa A3 that was isolated from the sample and was shown to have inhibitory activity towards pathogenic bacteria namely Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Micrococcus luteus. The bacteriocin was shown to be heat stable and functioned well at low pH (2 to 6). Reduction of activity was shown after treatment with proteinase K, trypsin and peptidase that confirmed the proteinaceous nature of the compound. MALDI-TOF analysis of the sample gave a mass approximating 2.7 kDa. The membrane of the bacteria was disrupted by the bacteriocin causing SYTOX^® green dye to enter the cell and bind to the bacterial DNA giving fluorescence signal. Bacterial cell treated with the bacteriocin also showed significant morphological changes under transmission electron microscope. No virulence and disease related genes can be detected from the genome of the strain.     

Purification and Characterization of a Novel Bacteriocin Produced by Enterococcus faecalis Strain RJ-11

Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.     

木霉菌发酵羊胃容物产木聚糖酶的分离纯化及特性

目的:为城市屠宰场牛羊胃容物类的生物垃圾型可再生资源的潜在工业化开发应用(城市无害鲜生物垃圾变废为宝的低碳生物循环经济工程)提供了部分可靠科学依据.方法:M-112木霉菌固态发酵羊胃容物的粗酶提取液,经20～60%饱和度的硫酸铵粗分级分离、Sephadex G-25脱盐、Sephadex G-150到Sephadex G-100分子筛顺序纯化,通过蛋白质测定、酶活测定、SDS-PAGE和底物-PAGE电泳等.结果:制备到一种分子量约为64.7kDa的电泳纯木聚糖酶,其回收率为5.59%,纯化倍数10.43,比活力为253.36IU/mg.纯化出的木聚糖酶,经性质研究表明,其Km=11.20g/L,Vmax=0.70μmoL/min;最适温度为65℃,最适pH为6.0,该酶在中温条件下稳定性好,在酸性至弱碱性(pH 3.0～8.0)条件下稳定性好.结论:采用改良的凝胶色谱技术,首次纯化出木霉菌固态发酵羊胃容物所产的木聚糖酶.