title: Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102.
abstract: There is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied.
  
  
  
  The best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 μg/mL.
  
  
  
  Taking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.
authors.affiliation: Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt. nouraelahmady@yahoo.com.
authors.forename: Noura El-Ahmady
authors.initials: NE
authors.lastname: El-Naggar
authors.affiliation: Department of Protein Research, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research & Technological Applications, Alexandria, Egypt.
authors.forename: Sahar F
authors.initials: SF
authors.lastname: Deraz
authors.affiliation: Department of Botany, Faculty of Science, Mansoura University, Mansoura, Egypt.
authors.forename: Hoda M
authors.initials: HM
authors.lastname: Soliman
authors.affiliation: Biopharmacetical Product Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt.
authors.forename: Nehal M
authors.initials: NM
authors.lastname: El-Deeb
authors.affiliation: Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt.
authors.forename: Nancy M
authors.initials: NM
authors.lastname: El-Shweihy
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D001426:Bacterial Proteins / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007700:Kinetics; D008970:Molecular Weight; D012685:Sepharose / Q000031:analogs & derivatives; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; C043982:DEAE-Sepharose CL-6B; D012685:Sepharose; D002789:Cholesterol Oxidase
keywords: Amino acid contents; Characterization; Cholesterol oxidase; DEAE Sepharose CL-6B; Molecular weight; Purification; Streptomyces aegyptia NEAE 102