Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent.
A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His6-tagging.
Department of Biochemistry, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
Karim Engelmark
KE
Cassimjee
Martin
M
Trummer
Cecilia
C
Branneby
Per
P
Berglund
D000411:Alanine Racemase / Q000737:chemistry*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry*; D045302:Geobacter / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D012822:Silicon Dioxide / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry
D004800:Enzymes, Immobilized; D012997:Solvents; D006639:Histidine; D012822:Silicon Dioxide; D000411:Alanine Racemase