A microreactor for the study of biotransformations by a cross-linked gamma-lactamase enzyme.
A (+)-gamma-lactamase was precipitated, cross-linked and the resulting solid crushed prior to immobilisation within a capillary column microreactor. The microreactor was subsequently used to study enzyme stability, activity, kinetics and substrate specificity. The thermophilic (+)-gamma-lactamase retained 100% of its initial activity at the assay temperature, 80 degrees C, for 6 h and retained 52% activity after 10 h, indicating the advantage of immobilisation. This high stability of the immobilised enzyme provided the advantage that it could be utilised to screen many compounds in the microreactor system. This advantage overcame the fact that the immobilisation process affected enzyme kinetics and activity, which was reduced (by 70%) compared to the free enzyme. In general, the enzyme displayed similar substrate specificity to that found in a previous study for the free enzyme; however, enhanced activity was seen towards one substrate, acrylamide. The system developed correlates well with the free enzyme in batch assay and indicates the suitability of the system for enzyme substrate screening, allowing a significant reduction in cost, due to the reduced amounts of enzyme, substrates and other assay constituents required.
Hickey
Anne Marie
AM
University of Exeter, UK.
Ngamsom
Bongkot
B
Wiles
Charlotte
C
Greenway
Gillian M
GM
Watts
Paul
P
Littlechild
Jennifer A
JA
D000581:Amidohydrolases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D019149:Bioreactors; D001711:Biotransformation; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D008904:Miniaturization; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D000581:Amidohydrolases; C401943:gamma-lactamase