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Study into the kinetic properties and surface attachment of a thermostable adenylate kinase.

Hathaway HJ, Sutton JM, Jenkins AT - Biochem Biophys Rep (2015)

Bottom Line: This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination.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.This needs to be taken into consideration when using the assay to estimate cleaning efficacy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Bath, Claverton Down, Bath and North East Somerset, BA2 7AY, UK.

ABSTRACT

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 (k cat/K M) increased from 8.45±3.04 mM(-1) s(-1) in solution to 32.23±3.20 or 24.46±4.41 mM(-1) s(-1) when 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.

No MeSH data available.


Comparison of initial rate of reaction (RLU s−1) when using a high tAK concentration (A) and for lower tAK concentrations (B) adsorbed to polypropylene. Tangents were fitted according to a linear fit function within Origin graphing software between 5 and 15 s.
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f0020: Comparison of initial rate of reaction (RLU s−1) when using a high tAK concentration (A) and for lower tAK concentrations (B) adsorbed to polypropylene. Tangents were fitted according to a linear fit function within Origin graphing software between 5 and 15 s.

Mentions: Different concentrations of tAK were immobilised under standard conditions (80% water, 20% ethanol, 0.1% mucin, reacted for 2 h at 37 °C with agitation) Data is shown as relative light units (Fig. 3). The initial rate of reaction was calculated between 5 and 15 s by means of tangents and plotted as a function of ADP concentration, as shown in Fig. 4.


Study into the kinetic properties and surface attachment of a thermostable adenylate kinase.

Hathaway HJ, Sutton JM, Jenkins AT - Biochem Biophys Rep (2015)

Comparison of initial rate of reaction (RLU s−1) when using a high tAK concentration (A) and for lower tAK concentrations (B) adsorbed to polypropylene. Tangents were fitted according to a linear fit function within Origin graphing software between 5 and 15 s.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4547157&req=5

f0020: Comparison of initial rate of reaction (RLU s−1) when using a high tAK concentration (A) and for lower tAK concentrations (B) adsorbed to polypropylene. Tangents were fitted according to a linear fit function within Origin graphing software between 5 and 15 s.
Mentions: Different concentrations of tAK were immobilised under standard conditions (80% water, 20% ethanol, 0.1% mucin, reacted for 2 h at 37 °C with agitation) Data is shown as relative light units (Fig. 3). The initial rate of reaction was calculated between 5 and 15 s by means of tangents and plotted as a function of ADP concentration, as shown in Fig. 4.

Bottom Line: This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination.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.This needs to be taken into consideration when using the assay to estimate cleaning efficacy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Bath, Claverton Down, Bath and North East Somerset, BA2 7AY, UK.

ABSTRACT

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 (k cat/K M) increased from 8.45±3.04 mM(-1) s(-1) in solution to 32.23±3.20 or 24.46±4.41 mM(-1) s(-1) when 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.

No MeSH data available.