• Title/Summary/Keyword: Purification and characterization

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Characterization of Aspartate Aminotransferase Purified from Streptomyces fradiae (Streptomyces fradiae에서 분리된 Aspartate Aminotransferase의 특성)

  • Lee, Sang-Hee;Lee, Kye-Joon
    • Korean Journal of Microbiology
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    • v.31 no.3
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    • pp.237-244
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    • 1993

  • Aspartate aminotransferase (ASAT) (L-aspartate : 2-oxyoglutarate, EC 2.6. 1. 1.) from Streptomyces fradiae NRRL 2702 has been purified by acetone precipitation, DEAE-cellulose, hydroxyapatite, and preparative electrophoresis (Prep cell), of which the last was the most effective step in the purification of ASAT. The molecular mass was estimated to be 54,000 dalton by SDS-PAGE and 120,000 dalton by gel filtration chromatography. Preparative isoelectric focusing of purified ASAT resulted in one polypeptide band with a pI of 4.2, showing homogeneity and indicating that the enzyme is composed of two identical subunits. The enzyme was specific for L-aspartate as an amino donor ; the $K_{m}$ values were determined to be 2.7 mM for L-aspartate, 0.7 mM for 2-oxoglutarate, 12.8 mM for L-glutamate, and 0.15 mM for oxaloacetate. The enzyme was relatively heat-stable, having maximum activity at 55.deg.C, and it had a broad pH optimum ranging from 5.5 to 8.0. The activity of the purified enzyme was not inhibited by ammonium ions. This paper reports the first purification and characterization of the aspartate aminotransferase from a species of Streptomyces.s.

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Purification and Characterization of Chloramphenicol Acetyltransferase from Morganella morganii

  • El-Gamal, Basiouny;Temsah, Samiha;Olama, Zakia;Mohamed, Amany;El-Sayed, Mohamed
    • BMB Reports
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    • v.34 no.5
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    • pp.415-420
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    • 2001

  • Chloramphenicol acetyltransferase (CAT) was purified to homogeneity from Morganella morganii starting with ammonium sulphate fractionation, followed by separation on DEAE-Sephadex A50, and G-100 Sephadex gel filtration. The enzyme was purified 133.3 fold and showed a final specific activity of 60 units/mg protein with a yield of 37%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it as a heterotetramer that consists of four subunits with close molecular weights (19.5, 19, 18, and 17.5 kDa). The molecular weight of the native enzyme was calculated to be 78 kDa, as determined by gel filtration, which approximated to that of the four subunits (74 kDa). The enzyme showed a maximum activity at pH 7.8 when incubated at $35^{\circ}C$. A Lineweaver-Burk analysis gave a Km of 5.0 uM and Vmax of 153.8 U/ml. The amino acid composition of the purified enzyme was also determined.

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Purification and Characterization of Recombinant Human Interferon Alpha 2a Produced from Saccharomyces cerevisiae

  • Rae, Tae-Ok;Chang, Ho-Jin;Kim, Jung-Ho;Park, Soon-Jae
    • BMB Reports
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    • v.28 no.6
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    • pp.477-483
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    • 1995

  • The recombinant human interferon alpha 2a ($rhIFN-{\alpha}2a$), expressed in Saccharomyces cerevtsiae, was purified from insoluble aggregates. The inclusion body of $rhIFN-{\alpha}$ was solubilized by guanidine salt in the presence of disulfide reducing agent. The refolding of denatured $rhIFN-{\alpha}2a$ was achieved by simple dilution. The authentic interferon alpha, which has two correctly matched disulfide bonds, was seperated from incompletely oxidized $IFN-{\alpha}$ and dimeric $IFN-{\alpha}$ by use of a CM-Sepharose column, followed by size exclusion columns at two different pH conditions. The purified protein has been subjected to detailed physicochemical characterization including sequence determination. Unlike other $rhIFN-{\alpha}2a$ from E. coli reported, the $rhIFN-{\alpha}2a$ from S. cerevisiae has no methionine residue at its N-terminus originating from the start codon, ATG. The pI of the protein was determined to be 6.05 with a single band in the pI gel, which demonstrated that the purified $rhIFN-{\alpha}$ was homogeneous. The structural study using circular dichroism showed that the protein retains its three dimensional structure in the wide range of pH conditions between pH 3 and 9, and only minor strucural deformation was observed at pH 1.0.

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