Stabilization of a formate dehydrogenase by covalent immobilization on highly activated glyoxyl-agarose supports.
Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor.
Bolivar
Juan M
JM
Departamento de Biocatalisis, Instituto de Catalisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Wilson
Lorena
L
Ferrarotti
Susana Alicia
SA
Fernandez-Lafuente
Roberto
R
Guisan
Jose M
JM
Mateo
Cesar
C
D001672:Biocompatible Materials / Q000737:chemistry; D002384:Catalysis; D003911:Dextrans / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005560:Formate Dehydrogenases / Q000737:chemistry*; D006038:Glyoxylates / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D020836:Protein Structure, Quaternary; D011549:Pseudomonas / Q000201:enzymology; D012685:Sepharose / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013696:Temperature
D001672:Biocompatible Materials; D003911:Dextrans; D004800:Enzymes, Immobilized; D006038:Glyoxylates; D046911:Macromolecular Substances; D012997:Solvents; D009243:NAD; C070399:glyoxyl agarose; D012685:Sepharose; D005560:Formate Dehydrogenases