Study into the kinetic properties and surface attachment of a thermostable adenylate kinase.
A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from8.45±3.04 mM-1s-1in solution to32.23±3.20 or24.46±4.41 mM-1s-1when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.
Department of Chemistry, University of Bath, Claverton Down, Bath and North East Somerset, BA2 7AY, UK.
H J
HJ
Hathaway
Technology Development Group, Public Health England, Porton Down, Salisbury, Wiltshire, UK.
J M
JM
Sutton
Department of Chemistry, University of Bath, Claverton Down, Bath and North East Somerset, BA2 7AY, UK.
A T A
AT
Jenkins
ADP, Adenosine Diphosphate; AK, Adenylate Kinase; AMP, Adenosine Monophosphate; ATP, Adenosine Triphosphate; CJD, Creutzfeldt –Jacob Disease; Cleaning efficacy; Enzyme inhibition; Kinetic parameters; Plasma deposition; RLU, Relative Light Units; Thermostable adenylate kinase; tAK, Thermostable Adenylate Kinase