Jin Cheol Yoo

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.

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 methicilin resistant Staphylococcus aureus (MRSA). Purified substance from the organism, CS-682, which is active against MRSA and Micrococcus leuteus, is a C28H37NO8 (M+H+, observed: 516.83) and identified as an unusual macrolide antibiotic, nargenicin. The chemical structure of CS-682 was identified by FT-IR, 1H-NMR, 13C-NMR, and (1H-1H and 1H-13H) 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 44704T (98.7% sequence similarity), followed by N. brasiliensis ATCC 19296T (98.4% sequence similarity). The ability of Nocardia sp. CS682 to produce nargenicin was unique.

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.

Nargenicin enhances 1,25-dihydroxyvitamin D3- and all-trans retinoic acid-induced leukemia cell differentiation via PKCβI/MAPK pathways

A major goal in the treatment of acute myeloid leukemia (AML) is to achieve terminal differentiation and prevent drug resistance and side effects. Combined treatment with low doses of ATRA or 1,25-(OH)(2)D(3) that do not induce toxicity with another drug is one useful strategy for the treatment of AML. Actinomycetes are the well known sources of antibiotics and bioactive molecules. Previously, we isolated nargenicin from the culture broth of an actinomycete isolate, Nocardia sp. CS682. In this study, we evaluated the effects of nargenicin on cellular differentiation in a human myeloid leukemia HL-60 cell system. Nargenicin inhibited cell proliferation and induced HL-60 cell differentiation when administered in combination with 1,25-(OH)(2)D(3) or ATRA. In addition, western blot analyses and kinase inhibitor studies demonstrated that nargenicin primarily enhanced leukemia cell differentiation via PKCbeta1/MAPK pathways. The results of this study indicate that nargenicin has the ability to induce differentiation and suggest that it may be useful for the treatment of neoplastic diseases.

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.

An organic solvent-tolerant alkaline lipase from Streptomyces sp. CS268 and its application in biodiesel production

In an effort to identify a microbial lipase that can catalyze transesterification reactions used in biodiesel production, an organic solvent-tolerant lipase was purified from Streptomyces sp. CS268. The molecular weight of the purified lipase was estimated to be 37.5 kDa by SDS-PAGE. The lipase showed highest activity at a temperature of 30°C and pH 8.0 while it was stable in the pH range 4.0 ∼ 9.0 and at temperatures ≤ 50°C. It showed the highest hydrolytic activity towards medium-length acyl chain p-nitrophenyl decanoate with Km and Vmax values of 0.59 mM and 319.5 mmol/mg/min, respectively. Also, the lipase showed non-position specificity for triolein hydrolysis. The purified lipase catalyzed transesterification reaction of soybean oil with methanol, suggesting that it can be a potential enzymatic catalyst for biodiesel production.

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.

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°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°C and 35°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°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.

Prevalence and characterization of vancomycin-resistant enterococci in chicken intestines and humans of Korea.

The prevalence, genotype for antibiotic resistance and antibiotic susceptibility of vancomycin resistant enterococci (VRE) were determined. And molecular typings of theEnterococcus faecium isolates were analyzed. Prevalence of VRE in chickens, healthy children and intensive care unit (ICU) patients was 41.6%, 7.9%, and 20.4%, respectively. Forty out of 54 isolates from chicken intestines, and 9 out of 11 from ICU patients were identified asEnterococcus faecium. Eleven out of 13 isolates from non-hospitalized young children wereE gallinarium. Twelve strains ofE faecalis were isolated from chicken intestines. The gene for the antibiotic resistance inE. faecium, andE. faecalis wasvanA, while that inE. gallinarium was vanC1. E. faecium isolates were resistant to most of antibiotics except ampicillin and gentamicin. Molecular typing of theE. faecium strains obtained by pulse field gel electrophoresis and repetitive sequence-based PCR suggest that VRE transmit horizontally from poultry to humans, especially young children,via the food chains in Korea.

Enhanced Production of Nargenicin A 1 and Generation of Novel Glycosylated Derivatives

Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-β- D-glucopyranoside, nargenicin A1 18-O-β-D-glucopyranoside, nargenicin A111 18-O-β-D- diglucopyranoside, and nargenicin 11-O-β-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-β-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.