Immobilization of deoxyribonuclease via epoxy groups of methacrylate monoliths. Use of deoxyribonuclease bioreactor in reverse transcription-polymerase chain reaction.
A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis-Menten constant, Km(app), and turnover number, k3app, for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28 g of DNA l(-1) and 16 dA260nm min(-1) mg(-1) of immobilized DNase. The highest activity of immobilized DNase was detected at 1 mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37 degrees C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR.
Bencina
Mojca
M
Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
Bencina
Katja
K
Strancar
Ales
A
Podgornik
Ales
A
D001483:Base Sequence; D019149:Bioreactors; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D007700:Kinetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004800:Enzymes, Immobilized; D003851:Deoxyribonucleases