Improving dioxygenase stability by gene chromosome insertion: implementation in immobilized-cell systems.
The immobilization of recombinant cells by using the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase was studied as a model. Dioxygenase activity and cell viability were compared in immobilized-cell systems and cells in suspension. Immobilization increased enzyme stability and the efficient degradation of 3,4-dihydroxyphenylacetate. The stability of the cloned enzyme and the viability of the immobilized recombinant cells were well maintained for at least 15 days. We used the strain Escherichia coli CC118-D in which the hpaB gene from Klebsiella pneumoniae, coding for the subunit of 3,4-dihydroxyphenylacetate 2,3-dioxygenase, was inserted into the chromosome. This study has demonstrated that the implementation of E. coli CC118-D in a pilot-scale bioreactor resulted in a 100% stabilization of dioxygenase activity, and could be a useful tool for bioremediation processes.
Gibello
A
A
Departamento Patologia Animal (Microbiologia), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
Garbi
C
C
Allende
J L
JL
Martin
M
M
D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D018914:Cells, Immobilized; D003001:Cloning, Molecular; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase