Jaya Ram Simkhada

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.

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.

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.

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 D3 [1,25-(OH)2D3] or all-trans retinoic acid (ATRA). Treatment of HL-60 cells with zero to 100 μg/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)2D3 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)2D3, 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)2D3 may improve the ultimate outcome of acute promyelocytic leukemia therapy.

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

An extracellular phospholipase D (PLDSt) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLDSt was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60°C, and the enzyme was stable at or below 60°C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca2+, and the maximum activity was at 2 mM CaCl2. The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 µmol min−1 mg−1, respectively. More importantly, PLDSt 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 PLDSt does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLDSt may therefore be a novel type of PLD enzyme.