title: [Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) and studying the enzymatic properties of its expression product].
abstract: The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21 (DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45 degrees C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15-20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45 degrees C and was completely inactivated after incubation at 65 degrees C for 1 h.
authors.forename: A S
authors.initials: AS
authors.lastname: Rozanov
authors.forename: S N
authors.initials: SN
authors.lastname: Zagrebel'nyĭ
authors.forename: A B
authors.initials: AB
authors.lastname: Beklemishchev
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D048168:Escherichia coli K12 / Q000201:enzymology*; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase