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Simkhada, Jaya Ram,Yoo, Hah-Young,Choi, Yun Hee,Kim, Seung Wook,Yoo, Jin Cheol Humana Press 2012 Applied biochemistry and biotechnology Vol.168 No.7
<P>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.</P>
Purification and Biochemical Properties of Phospholipase D (PLD57) from Streptomyces sp. CS-57
Jaya Ram Simkhada,Seung Sik Cho,하정완,유진철 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.10
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 28oC, 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 45oC and 35oC, 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 45oC 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 New Thermolabile Alkaline Phospholipase D from Streptomyces sp. CS628
Simkhada, Jaya Ram,Cho, Seung-Sik,Choi, Hong-Seok,Kim, Si-Wouk,Lee, Hei-Chan,Sohng, Jae-Kyung,Yoo, Jin-Cheol 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.4
A phospholipase D ($PLD_{628}$), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at $30^{\circ}C$ with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, $30^{\circ}C$, pH 5.7 ~ 10.6 and ${\leq}30^{\circ}C$, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, $PLD_{628}$ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that $PLD_{628}$ lacks the transphosphatidylation activity. $PLD_{628}$ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.
Simkhada, Jaya Ram,Cho, Seung-Sik,Lee, Hyo-Jung,Yoo, Jin-Cheol 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.10
Streptomyces sp. CS-57, which was isolated from Korean soil, was found to produce phospholipase D ($PLD_{57}$) as an extracellular enzyme when cultured in medium containing 2% glucose, 1.5% yeast extract, 0.5% trypton, and 0.1% calcium carbonate at $28^{\circ}C$, and 160-rpm. $PLD_{57}$ 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. $PLD_{57}$ showed both hydrolytic (H) and transphosphatidylation (T) activity, and the optimum temperatures of these activities were found to be $45^{\circ}C\;and\;35^{\circ}C$, 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 $45^{\circ}C$ 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.
Monoclonal Antibody Production and Immunochemical Detection of Polyether Antibiotics
이효정,조승식,Jaya Ram Simkhada,유진철 대한약학회 2009 Archives of Pharmacal Research Vol.32 No.3
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).