Bioimprinting of immobilized Lecitase via physical
coating with polymers in the presence of
detergents
J.C.S. Santos1,2, N. Rueda1,3, Oveimar Barbosa3, C. Garcia-Galan1, L.R.B.
Gonçalves2, R.C. Rodrigues5, and R. Fernandez-Lafuente1*
1Instituto de Catálisis-CSIC, Campus UAM-CSIC, Cantoblanco, Madrid, Spain; 2Departamento de Engenharia Química, Universidade
Federal do Ceará, Fortaleza, CE, Brazil; 3Universidad Industrial de Santander, Bucaramanga, Colombia. 4Federal University of Rio
Grande do Sul, Porto Alegre, RS, Brazil. 5Instituto de Química Avanzada de Cataluña-CSIC Jordi Girona 18-26, 08034. Barcelona
(Spain. *[email protected]
Keywords: Enzyme hyperactivation, Detergents, Solid-phase physical modification.
INTRODUCTION
Lipase activity in aqueous media is reduced
due to the opening/closing equilibrium. Detergents
may stabilize the open form of lipase, improving its
activity. However, they can present some
detrimental effects on enzyme stability. Coating of
lipases with ionic polymers in the presence of
detergents is here proposed as a tool to improve
lipase performance.
RESULTS AND DISCUSSION
negative effect on enzyme stability even at low
concentrations (0.01 % (v/v)) and neither SDS can
be used for long times at 1 % concentration. In order
to keep the hyperactivated form of the enzyme in the
absence of detergent (that is deleterious for the
stability), ionic polymers were added during the
incubation of the immobilized enzyme in different
concentrations
of
detergents.
Coating
the
immobilized enzyme with polyethylenimine (PEI)
produced a 3-fold increase in enzyme activity
1
(Figure 1) . However, in the presence of 0.1 % SDS
(v/v), this PEI coating produced a 50-fold increase in
enzyme activity2. Using an irreversible inhibitor, the
inhibition rate showed that the PEI/SDS-CNBrLecitase preparation presented its catalytic Ser more
exposed to the reaction medium than the unmodified
CNBr-Lecitase.
CONCLUSION
Figure 1. Effect of SDS concentration and incubation time on the
activity of immobilized Lecitase in presence or absence of PEI.
Dashed line, empty squares: CNBr-Lecitase incubated with 0.1%
SDS, rhombus: CNBr-Lecitase incubated with 0.002% SDS and
PEI, circles: CNBr-Lecitase incubated with 0.01% SDS and PEI,
triangles: CNBr-Lecitase incubated with 0.05% SDS and PEI,
squares: CNBr-Lecitase incubated with 0.1% SDS and PEI.
Lecitase Ultra, a phospholipase A1, was
covalently immobilized on Cyanogen bromide crosslinked 4 % agarose (CNBr) beads, maintaining 70%
of the initial activity. The activity of the immobilized
enzyme, Lecitase-CNBr, in the presence of
detergents, such as Triton X-100, sodium dodecyl
sulphate (SDS), cetyltrimethyl ammonium bromide
(CTAB) increased, up to 800 % when using CTAB.
However, CTAB and Triton-X100 presented a
The coating of immobilized Lecitase Ultra
with PEI in the presence of SDS permits the
stabilization of the open form of the enzyme,
increasing th activity of the immobilized enzyme
even by a 50 fold factor.
ACKNOWLEDGEMENTS
We gratefully the Spanish Government,
grant CTQ2009-07568 and CTQ2013-41507-R and
CNPq (Brazil) for financial support. The authors wish
to thank Ramiro Martínez (Novozymes, Spain) for
kindly supplying the enzymes used in this research.
REFERENCES
[1] Santos, JCS; Garcia-Galan, C; Rodrigues, RC; Ana HBS;
Goncalves,LRB; Fernandez-Lafuente, R. Enzyme Microb Technol
2014;60:1–8.
[2] Santos, JCS; Garcia-Galan, C; Rodrigues, RC; Ana HBS;
Goncalves,LRB; Fernandez-Lafuente, R. Process Biochem DOI:
10.1016/j.procbio.2014.05.009.
VII Workshop on Biocatalysis and Biotransformations and 1o Simposio Latinoamericano de Biocatalisis y Biotransformaciones
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Bioimprinting of immobilized Lecitase via physical coating with