Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-Sepharose.
The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an 'affinity tag' to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.
Persson
M
M
Pure and Applied Biochemistry, Chemical Center, University of Lund, Sweden.
Bülow
L
L
Mosbach
K
K
D000345:Affinity Labels; D002846:Chromatography, Affinity; D003545:Cysteine; D004220:Disulfides; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D006358:Hot Temperature; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives
D000345:Affinity Labels; D004220:Disulfides; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C035872:thiopropyl-sepharose; D012685:Sepharose; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D003545:Cysteine