Monitoring the Activity of Immobilized Lipase with Quinizarin Diester Fluoro-Chromogenic Probe

• Published in: Molecules (Basel, Switzerland) Switzerland), 2017-12, Vol.22 (12), p.2136
• Main Authors: Aparecida Sabatini, Carolina, Massucatto Dos Santos, Denis, Matos de Oliveira da Silva, Sabrina, Henrique Gehlen, Marcelo
• Format: Article
• Language: English
• Subjects: Anthraquinones - chemistry; Biocatalysts; Enzymatic activity; Enzyme activity; Enzyme kinetics; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Fluorescence; fluorochromic substrate; Lipase; Lipase - chemistry; Lipase - metabolism; lipase assay; Organic solvents; Quinizarin; quinizarin diester; Reaction time; Spectrum analysis
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules22122136

Quinizarin diester is used as a fluoro-chromogenic substrate of the activity of lipase supported in poly(methylmetacrylate) beads (CALB, Novozym 435) dispersed in organic solvents. The monoester and diester of quinizarin are both non-fluorescent species contrasting with the enzymatic product quinizarin that shows optical absorption in the visible region and strong fluorescence signal. The enzymatic conversion is accomplished by spectroscopic measurements and it follows a sigmoid curve from which the mean reaction time of the enzymatic process can be determined. This parameter indicates the enzyme activity of the immobilized lipase. Its dependency with the amount of lipase allowed the determination of the ratio of the catalytic rate and the Michaelis constant ( / ) and the experimental value found was (1.0 ± 0.1) × 10 mg /min in the case of quinizarin diacetate.