Analysis of proteolytic degradation of a crude protein mixture using a surface acoustic wave sensor.
Degradation of a crude protein mixture by proteases with pH optima from acidic to basic was followed in real time using a surface acoustic wave biosensor in Love-wave geometry. Proteases EC 3.4.23.18 from Aspergillus saitoi, EC 3.4.21.62 from Bacillus licheniformis, and Novozyme from Bacillus sp. have been used. Kinetic constants extracted from the curves resulted for comparable protease concentrations in maximal degradation rates between 1.1 x 10(-2) and 1.5 x 10(-2)s(-1). For the three proteases investigated, similar amounts of up to about two-thirds of the estimated 28 ng/cm2 bound molecules were proteolyzed. The residual material not degraded by the proteases was removed from the surface with 0.5% SDS. The analysis of the sensor signal allows: (1) estimation of the total mass of protein bound to the sensor surface and of the degradable fraction; (2) extraction of the pure mass signal; and (3) kinetic evaluation.
Gronewold
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Center of Advanced European Studies and Research, S-sens, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany. gronewold@caesar.de
Schlecht
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Quandt
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D015374:Biosensing Techniques / Q000295:instrumentation*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000502:physiology*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
D011506:Proteins; D010447:Peptide Hydrolases