3rd meeting of young researchers at UP 1 - IJUP - Universidade do ...

Automated evaluation of the effect of ionic liquids on immobilized
trypsin
Susana P.F. Costa, Marieta L.C. Passos, M. Lúcia M.F.S. Saraiva, Paula C.A.G. Pinto,
José L.F.C. Lima
REQUIMTE/Department of Physical-Chemistry, Faculty of Pharmacy, University of Porto, Porto
The increasing use of ionic liquids in biocatalytic procedures with interesting results in terms
of sensitivity and selectivity demands specific studies of enzyme activity to evaluate the effect
of these solvents on enzyme behavior [1]. Usually, this kind of studies is performed on
enzymes in solution but the utilization of immobilized biocatalysts seems to be a good option
for routine implementation since it guarantees good catalytic activity and enzyme reutilization,
leading to a significant reduction of analysis cost [2]. In this work the enzyme trypsin was
immobilized in alkyl amine glass beads using glutaraldehyde as cross-linking reagent. The
immobilized enzyme was introduced in a perspex column (i.d.: 4 mm; 3 cm) and the reactor
was incorporated in a sequential injection analysis (SIA) system enabling the easy automation
of the procedure for enzyme activity evaluation.
The methodology was based on the hydrolysis of Nα-benzoyl-DL-arginine-4-nitroanilide
(BAPNA) resulting in the release of 4-nitroaniline whose absorbance was monitored at 405
nm. This kind of substrate has been used in enzyme activity assays resulting in simple and
effective methodologies [3].
In each cycle 50 µL of BAPNA were aspirated to the holding coil at a flow rate of 3 mL min -1
and sent, by flow reversal, to the enzyme reactor. Then, a stop period of 90 s was implemented
and after that the reaction product was sent to the detector at a flow rate of 1.5 mL min -1 . A
signal proportional to the enzyme activity was registered.
In the assays in ionic liquids, BAPNA was prepared in solutions of bmim [BF4] (1-butyl-3methyl-imidazolium
tetrafluoroborate), emim [BF4] (1-ethyl-3-methylimida-zolium
tetrafluoroborate and emim [Tf2] (1-ethyl-3-methylimidazolium trifluormethane-sulfonate) up
to 50% in order to investigate the effect of these ionic liquids on immobilized trypsin activity.
Acknowledgments: Susana Costa thanks for a grant in the scope of the project
BII/REQUIMTE/GABAI-6/2009
References:
[1] Kragl, U., Eckstein, M. and Kaftzik, K. (2002), Enzyme catalysis in ionic liquids, Curr. Opin.
Biotechnol., 13, 565-571.
[2] Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M. and Fernandez-Lafuente, R. (2007)
Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enz. Microb.
Technol., 40, 1451-1463.
[3] Nakashima, K., Maruyama, T., Kamiya, N. and Goto, M. (2008), Spectrophotometric assay for
protease activity in ionic liquids using chromogenic substrates, Anal. Biochem., 374, 285-290.
332 3 rd meeting of young researchers at UP