Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilized on nanosponges.
Catechol 1,2-dioxygenases are iron containing enzymes able to convert catechol into cis,cis-muconate, a precursor of the industrially important compound adipic acid. Catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 was immobilized on beta-cyclodextrins cross-linked with carbonate groups (nanosponges) with a yield of 29 mg of enzyme per gram of support. This support was chosen for its low cost and its ability to offer different types of interactions with the enzyme. The activity profiles at different pH and temperatures showed a shift of the optimal pH from 8.5, for the free protein, to 9.5, for the immobilized protein and, similarly, a shift in optimal temperature from 30 degrees C to 50 degrees C. The Michaelis-Menten constant, KM, increased from 2.0 +/- 0.3 microM, for the free form, to 16.6 +/- 4.8 microM for the immobilized enzyme, whereas the rate constant, k(cat), values were found to be 32 +/- 2 s(-1) and 27 +/- 3 s(-1) for the free and immobilized forms respectively. The immobilization process also increased the thermostability of the enzyme with 60% residual activity after 90 min at 40 degrees C for the immobilized protein versus 20% for the free enzyme. A residual activity of 75% was found after 15 min at 60 degrees C for the immobilized enzyme while the free form showed a total loss of activity under the same conditions. The activity toward other substrates, such as 3- and 4-methylcatechol and 4-chlorocatechol, was retained by the immobilized enzyme. A small scale bioreactor was constructed and was able to convert catechol into cis,cis-muconic acid with high efficiency for 70 days.
Di Nardo
Giovanna
G
Department of Human and Animal Biology, University of Torino, via Accademia Albertina 12, 10123, Torino, Italy.
Roggero
Carlo
C
Campolongo
Simona
S
Valetti
Francesca
F
Trotta
Francesco
F
Gilardi
Gianfranco
G
D000150:Acinetobacter / Q000201:enzymology*; D055162:Biocatalysis; D050548:Catechol 1,2-Dioxygenase / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049329:Nanostructures / Q000737:chemistry*; D013011:Sorbic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D047392:beta-Cyclodextrins / Q000737:chemistry
D002396:Catechols; D004800:Enzymes, Immobilized; D047392:beta-Cyclodextrins; C005939:muconic acid; D050548:Catechol 1,2-Dioxygenase; C034221:catechol; D013011:Sorbic Acid