A novel multifunctional peptide oligomer of bacitracin with possible bioindustrial and therapeutic applications from a Korean food-source Bacillus strain.

CONCLUSION: CSP32 has stable characteristics and may find bio-industrial and therapeutic applications.

An ammonium sulfate sensitive endoxylanase produced by Streptomyces.

Streptomyces sp. CSWu2 was newly isolated and identified from Korean soil. In culture medium, the strain produced a highly active endoxylanase (Xynwu2), which was purified to homogeneity by a single-step chromatography on Poros-HQ. The xylanase was ~38 kDa and its activity was maximal at 65 °C and pH 11.0. It was stable up to 60 °C and from pH 8.0 to 12.0, and its activity was slightly enhanced by nonionic detergents, but inhibited by EDTA, EGTA, and divalent metal ions. Intriguingly, Xynwu2 was highly sensitive to ammonium sulfate, but its completely suppressed activity was recovered by desalting out. Xynwu2 produced xylose and xylobiose as principal end products from xylan, suggesting an endoxylanase nature. Importantly, scanning electron microscopy showed Xynwu2 efficiently degraded corncobs, an agro-industrial waste material. We believe that Xynwu2 is a potential candidate for converting lignocellulosic waste material into simple sugars which could be used to produce bioethanol and other value-added products.

An extremely alkaline novel xylanase from a newly isolated Streptomyces strain cultivated in corncob medium.

Streptomyces sp. CS802, recently isolated from Korean soil, produced xylanase in corncob medium. An extracellular xylanase (Xyn802) was purified by a single-step gel filtration and biochemical properties were studied. It showed high activity in extremely alkaline condition with optimum pH at 12.0 and exhibited stability between pH 7.5 and 13.0. It produced xylobiose and xylotriose as the major products from xylan, suggesting its endoxylanase nature. N-terminal amino acid sequences of Xyn802 were ADRNANRD which are significantly different from the reported xylanase. The activity was enhanced by various detergents and a reducing agent and stable in various organic solvents. Xyn802 produced by utilizing corncob, an agro-waste material, might be a novel xylanase based on its peculiar biochemical characteristics, and it can be a suitable candidate for the production of xylooligosaccharides including other useful products.

A novel thermotolerant and acidotolerant peptide produced by a Bacillus strain newly isolated from a fermented food (kimchi) shows activity against multidrug-resistant bacteria.

In an attempt to isolate effective antimicrobial peptides (AMPs) from a microbial source for the treatment of multidrug-resistant (MDR) bacteria, BCP61 was purified from Bacillus sp. CS61 newly isolated from the traditional fermented food kimchi. BCP61 (ca. 1100 Da) was purified to homogeneity using sequential chromatographic steps. It was found to be stable at pH 2.0-10.0 and up to 80 °C. BCP61 displayed antimicrobial activity against MDR bacteria such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant enterococci (VRE). Minimal inhibitory concentrations of BCP61 for MRSA, VRSA and VRE were in the range 0.625-20µg/mL. The N-terminal amino acid sequence of BCP61 was A-I-N-X-D-A-A-Y-L, which differed from reported AMPs. The fourth unidentified amino acid was replaced and several peptides were synthesised. Among them, only cysteine replacement displayed antimicrobial activity. BCP61 from a food-borne strain may be useful in therapeutic applications.

Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model.

INTRODUCTION: The prevalence of cardiovascular diseases, one of the major causes of worldwide mortality, is being increasingly reported. Safer, more effective, and less expensive thrombolytic drugs can possibly overcome the underlying problems associate with current thrombolytic drugs. METHODS: A thrombolytic enzyme was purified and characterized from a Streptomyces strain. Carrageenan induced tail-thrombosis mice model was used to evaluate in vivo antithrombotic effect of the enzyme. RESULTS: First 15N-terminal amino acids of the purified enzyme were IAGGQAIYAGGGRRS, which are significantly different from the reported fibrinolytic enzymes. The enzyme exhibited 14.3±2.3-fold stronger thrombolytic activity than that of plasmin. In carrageenan induced tail-thrombosis model, the enzyme caused reduction in frequency of thrombus. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. CONCLUSIONS: The enzyme purified from the Streptomyces strain can be a potential candidate for the treatment of thrombosis.

A newly isolated Streptomyces sp. CS392 producing three antimicrobial compounds.

With the aim of isolating new microbes capable of producing strong antimicrobial substances, strain CS392 was screened from 700 soil isolates preserved in our laboratory. The strain was related to genus Streptomyces based on various characteristics. Three highly active antimicrobial compounds, C1, C2 and C3, produced by the strain were purified by solvent extraction followed by silica gel column chromatography. These compounds were highly active against various Gram-positive resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococcus (VRE). Among three, C3 was the most active against MRSA and VRSA with minimal inhibitory concentration (MIC) of 2 µg/ml while C2 and C3 had MIC values of 4 µg/ml for the strains. In case of Bacillus subtilis ATCC6633, C1 and C3 were more effective with MIC values of 0.5 µg/ml than C2 with MIC of 2 µg/ml. Those antibiotics were variably active (MIC of 4-32 µg/ml) against Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Mycobacterium smegmatis ATCC 9341 and VRE.

A neutral lipase applicable in biodiesel production from a newly isolated Streptomyces sp. CS326.

In an attempt to isolate a biocatalyst able to catalyze biodiesel production from microbial source, Streptomyces sp. CS326 was screened from hundreds of soil isolates collected from various parts of Korea. In 16S rRNA sequence analysis, the strain showed high degree of similarity with Streptomyces xanthocidicus (99.79%); therefore, it is classified as Streptomyces sp. CS326. An extracellular lipase produced by the strain (LP326) was purified using a single step gel permeation chromatography on Sepharose CL-6B. Molecular weight of LP326 was estimated to be 17,000 Da by SDS-PAGE. The activity was optimum at 40 °C and pH 7.0 and was stable at pH 5.0-8.0 and below 50 °C. It preferred p-nitrophenyl palmitate (C16), a long chain substrate; and K (m) and V (max) for the substrate were determined to be 0.24 mM and 4.6 mM/min mg, respectively. First 10 N-terminal amino acid sequences were APDLVALQSE, which are different from so far reported lipases. LP326 catalyzed biodiesel production using methanol and various oils; therefore, the enzyme can be applicable in the field of biofuel.

A novel cold-adapted lipase, LP28, from a mesophilic Streptomyces strain.

Fossil fuel is limited but its usage has been growing rapidly, thus the fuel is predicted to be completely running out and causing an unbearable global energy crisis in the near future. To solve this potential crisis, incorporating with increasing environmental concerns, significant attentions have been given to biofuel production in the recent years. With the aim of isolating a microbial biocatalyst with potential application in the field of biofuel, a lipase from Streptomyces sp. CS628, LP28, was purified using hydroxyapatite column chromatography followed by a gel filtration. Molecular weight of LP28 was estimated to be 32,400 Da by SDS-PAGE. The activity was the highest at 30 °C and pH 8.0 and was stable at pH 6.0-8.0 and below 25 °C. The enzyme preferentially hydrolyzed p-nitrophenyl decanoate (C10), a medium chain substrate. Furthermore, LP28 non-specifically hydrolyzed triolein releasing both 1,2- and 1,3-diolein. More importantly, LP28 manifestly catalyzed biodiesel production using palm oil and methanol; therefore, it can be a potential candidate in the field of biofuel.

A novel alkaline lipase from Ralstonia with potential application in biodiesel production.

With the aim of isolating a biocatalyst able to catalyze biodiesel production from microbial source, Ralstonia sp. CS274 was isolated and a lipase from the strain (RL74) was purified. Molecular weight of RL74 was estimated to be 28,000 Da by SDS-PAGE. The activity was highest at 50-55°C and pH 8.0-9.5 and was stable at pH 7.0-12.0 and up to 45°C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and K(m) and V(max) for p-nitrophenyl palmitate were 2.73 ± 0.6mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.

An oxidant- and organic solvent-resistant alkaline metalloprotease from Streptomyces olivochromogenes.

Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the enzyme was estimated to be 51 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gelatin zymography. The activity was optimum at pH 7.5 and 50 degrees C and stable between pH 7.0 and 10.0. SOMP was stable below 45 degrees C and Ca(2+) increased its thermostability. Ca(2+) enhanced while Co(2+), Cu(2+), Zn(2+), Mn(2+), and Fe(2+) inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity, suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents, and oxidizing agents, and the activity is enhanced by H(2)O(2). The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological applications especially for organic solvent-based enzymatic synthesis.

Effects and action mechanism of Diospyros kaki on the differentiation of human leukemia HL-60 cells.

Diospyros kaki Thunb. (Ebenaceae) is widely distributed in North-East Asian countries. Almost all parts of this plant have been traditionally used as medicine. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or all-trans retinoic acid (ATRA). Combination of low doses of ATRA or 1,25-dihydroxyvitamin D3 that do not induce toxicity with another drug is a useful strategy for acute promyelocytic leukemia therapy. Our main aim was to investigate the effect of an acetone extract of D. kaki leaves (KV-1) on HL-60 cell differentiation in combination of ATRA or 1,25-dihydroxyvitamin D3. Treatment of HL-60 cells with zero to 100 microg/ml of KV-1 for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when the HL-60 cells were treated with ATRA or 1,25-(OH)2D3 and the extract. The inhibitors of protein kinase C (PKC) (alpha and betaI) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K) and c-Jun N-terminal kinase (JNK) inhibited the HL-60 differentiation induced by the extract in combination of ATRA or 1,25-(OH)2D3, suggesting that PKC and ERK were involved in the cell differentiation enhancement by the extract. The results indicate that the acetone extract of D. kaki leaves has the ability to enhance HL-60 cell differentiation and suggest that it may be useful in acute promyelocytic leukemia therapy.

A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity.

An extracellular phospholipase D (PLD(St)) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD(St) was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca(2+), and the maximum activity was at 2 mM CaCl(2). The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 micromol min(-1) mg(-1), respectively. More importantly, PLD(St) could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD( St ) does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD(St) may therefore be a novel type of PLD enzyme.

Enhancement of 1,25-dihydroxyvitamin D3- and all-trans retinoic acid-induced HL-60 leukemia cell differentiation by Panax ginseng.

Ginseng (Panax ginseng C.A. Meyer) has a wide range of therapeutic uses including cancer treatment. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Treatment of HL-60 cells with zero to 100 microg/ml of a methanol extract of ginseng for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when HL-60 cells were treated with ATRA or 1,25-(OH)(2)D(3) and the extract. The inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K), inhibited the HL-60 differentiation induced by the extract in combination with ATRA or 1,25-(OH)(2)D(3), signifying that PKC and ERK were involved in the cell differentiation enhancement by the extract. These results suggest that the ability of a methanol extract of ginseng to enhance the differentiation potential of ATRA or 1,25-(OH)(2)D(3) may improve the ultimate outcome of acute promyelocytic leukemia therapy.

Quantitative analysis of nargenicin in Nocardia sp. CS682 culture by high performance liquid chromatography.

A simple and sensitive HPLC method was developed for the quantitative analysis of nargenicin in the culture broth of Nocardia sp. CS682, isolated from Korean soil. Nargenicin, an unusual macrolide antibiotic with potent anti-MRSA (methicilin-resistant Staphylococcus aureus) activity, was extracted from the culture broth with ethylacetate and then analyzed by HPLC using solvent A (0.5 M dipotassium phosphate pH 2.5-water: acetonitrile = 80:20, v/v) and solvent B (100% acetonitrile), at 215 nm. The calibration curve of nargenicin was linear with correlation coefficient greater than 0.99. This method provides precise analysis of the nargenicin amount present in the cell free culture medium, and may be applicable to analyze other bacterial cultures containing similar compounds.

Monoclonal antibody production and immunochemical detection of polyether antibiotics.

Polyether antibiotics such as monensin and salinomycin have been primarily used as coccidiostat and growth promoter. Since residues of these antibiotic in food may pose a health risk for sensitive individuals, their use should be carefully monitored. An immunochemical method was developed for the determination of polyether antibiotic using monoclonal antibody (Mab) produced by immunized mice. Conjugates of monensin, salinomycin and laidlomycin were prepared with bovine serum albumin (BSA), keyhole limpet haemocyanine (KLH) and ovalbumin (OVA) by mixed anhydride method and then used as immunogene to produce Mab. Eight hybridoma cell lines were isolated that produced Mabs that competed with polyether antibiotic-protein conjugates in BALB/c-SP2/0 fusion system. Two hybridoma with higher sensitivity, designated as 4G11F and 1C8F1F, were cultured for mass production and then purified from ascites fluid. Antibiotic-protein conjugates were quantitavely analyzed by using the purified Mabs through a competitive enzyme-linked immunosorbent assay (ELISA).

A novel alkalo- and thermostable phospholipase D from Streptomyces olivochromogenes.

A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75 degrees C and the enzyme was stable from pH 7 to 13 and from 55 to 75 degrees C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca(2+)-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 mumol min(-1) mg(-1), respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50 degrees C. The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry.

A novel low molecular weight phospholipase D from Streptomyces sp. CS684.

With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids.

Production, isolation and biological activity of nargenicin from Nocardia sp. CS682.

Culture broth of an actinomycete isolate, Nocardia sp. CS682 showed specifically higher antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA). Purified substance from the organism, CS-682, which is active against MRSA and Micrococcus leuteus, is a C(28)H(37)NO(8) (M+H(+), observed: 516.83) and identified as an unusual macrolide antibiotic, nargenicin. The chemical structure of CS-682 was identified by FT-IR, (1)H-NMR, (13)C-NMR, and ((1)H-(1)H and (1)H-(13)H) COSY. The anti-MRSA activity of CS-682 was stronger than that of oxacillin, vancomycin, monensin, erythromycin, and spiramycin. Phylogenetic analysis showed that strain CS682 is closely related to Nocardia tenerifensis DSM 44704(T) (98.7% sequence similarity), followed by N. brasiliensis ATCC 19296(T) (98.4% sequence similarity). The ability of Nocardia sp. CS682 to produce nargenicin was unique.

Purification and biochemical properties of phospholipase D (PLD57) produced by Streptomyces sp. CS-57.

Streptomyces sp. CS-57, which was isolated from Korean soil, was found to produce phospholipase D (PLD57) as an extracellular enzyme when cultured in medium containing 2% glucose, 1.5% yeast extract, 0.5% trypton, and 0.1% calcium carbonate at 28 degrees C, and 160-rpm. PLD57 was purified using Sepharose CL-6B column chromatography, and DEAE-Sepharose CL-6B ion exchange column chromatography. The specific activity of the purified enzyme increased 6.7 fold with 3% recovery. The purified enzyme was then analyzed using 12% SDS-PAGE, which revealed that the molecular mass of the purified enzyme was 55 kDa. PLD57 showed both hydrolytic (H) and transphosphatidylation (T) activity, and the optimum temperatures of these activities were found to be 45 degrees C and 35 degrees C, respectively. Similarly, both of these activities were found to be optimal at a pH of 7.5. In addition, even after being heat treated at 45 degrees C for up to 2 h, the enzyme activity remained at 100%, and the H-activity was found to be stable at a pH of 6 to 8. Further, enzyme activity occurred in the presence of EDTA, indicating that metal ions are not required for their activity, although some metal ions did marginally increase the activity. Enzyme activity also increased by 75% in the presence of Triton-X 100 at a concentration of 0.375 %; however, none of the other detergents evaluated in this study were found to enhance enzyme activity.