Immobilisation of suitable mutA biochemical analysis of the secretory non-specific nuclease from S. Marcescens as a biotechnological tool

Project Information

Grant agreement ID: INTAS-94-1181

Project closed

Start date 1 August 1995

End date 31 July 1998

Funded under

• International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993-

Total cost

No data

EU contribution

€ 30 000,00

Coordinated by

Justus Liebig Universität Giessen
Germany

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Exploitable results

Immobilisation of suitable mutA biochemical analysis of the secretory non-specific nuclease from S Marcescens as a biotechnological tool

The Serratia nuclease is one of the most active endonucleases known and one of the very few that is active against all types of nucleic acids, namely single-and double-stranded RNA and DNA. This makes this nuclease an ideal enzyme to remove nucleic acids from biochemical and pharmaceutical preparations, as well as to quantitatively measure the nucleic acid content in such preparations by a potentiometric measurement. It would be advantageous for these purposes to have the Serratia nuclease covalently bound to a solid support, e.g. membranes, beads or microtitre-plate surfaces. A prerequisite for immobilization at high yield and with full preservation of activity is that the coupling involves a defined residue far away from the active site, such that the immobilization does not interfere with substrate binding and cleavage as well as that there are no adverse effects on the stability of the protein. For this purpose the active site of the Serratia nuclease was located by a mutational analysis the mechanism of action of this nuclease unravelled by a combination of mutational analysis and kinetic measurements and substrate preferences identified. Using a combination of chemical and biochemical techniques obligatory monomeric variants and covalently crosslinked dimeric variants were produced which should be better suited than the wild type enzyme for immobilization on solid support. Various biochemical constructs, protocols and matrices were investigated for their suitability to produce a Serratia nuclease variant immobilized on solid support with high yield, activity and stability. This part of the work ist not quite complete but the general strategy has been established.