DEXTRANSUCRASE ENTRAPMENT AS AN EFFICIENT ALTERNATIVE FOR INCREASED RECYCLING

EFFICIENCY OF FREE ENZYME WITHIN AGAR-AGAR FILM MATRIX

Miona Miljkovića, Slađana Davidovića, Neda Radovanovićb, Milan Gordićc, Milica Carevića, Aleksandra Nešićc,d, Suzana Dimitrijevića
aUniversity
of Belgrade, Faculty of Technology and Metallurgy, Serbia
bUniversity of Belgrade, Innovation Center of Faculty of Technology and Metallurgy, Serbia
cUniversity of Belgrade, Vinča Institute of Nuclear Sciences, Serbia
dUniversity of Concepción, Technological Development Unit, Coronel, Correo 3, Concepción, Chile

Dextransucrase (DS), the extracellular enzyme is of immense industrial importance, due to ability to produce dextran and oligosaccharides (OS). Worldwide
interest in OS has been increasing, since they have been accorded the prebiotic status. However, the industrial application of DS for OS synthesis is limited, due
to low yield of enzyme production and its low catalytic activity. Hence, there is a great interest for development of new technologies that can provide
improved performance of biocatalyst. Enzyme immobilization technology is considered to be a crucial step for cheaper and more efficient usage of DS.
Entrapment is one of the widely investigated immobilization methods, where enzymes are enclosed or confined within the polymer matrix without altering
their native structure, developing bioreactors for commercial applications. Different matrices such as polyacryl-amide gel, alginate beads and agar–agar have
been used for the entrapment of different enzymes and among them agar–agar is a biocompatible, non-toxic and strong solidifying agent for immobilization of
various enzymes.

Composition • Mechanical properties:

2, 2.5 and 4 wt tensile strength, elongation
Sample Agar-agar DS
% agar – agar (ml) (ml) at break and elastic
glycerol modulus
DS Control 7 (2%*) 0
9:1 6.3 (2%*) 0.7
• Enzymatic activity and
After cooling
After drying reusability
to 40-45oC 4:1 5.6 (2.5%*) 1.4
1:1 3.5 (4%*) 3.5
• HPLC analysis
T = 100 °C *percent of agar-agar mixed with DS
• SEM analysis

OS yield production by immobilized (1:9) and free

Mechanical properties of agar-agar and agar-agar with entrapped DS dextransucrase after 24h is presented in chromatogram
films are presented in Table 1: (Figure 1.):
Thickness, mm Speed, mm/min σ, MPa E, MPa Elongation at break, %
control 0.050 5 23.745 343.423 15.327
1:9 0.045 5 26.637 553.935 12.56
1:4 0.045 5 19.996 439.624 10.35
1:1 0.045 5 † † †

† samples were not uniform so that measurements are not accurate

The lowest tested fraction of enzyme immobilized into polymer matrix

(1:9) improved tensile strength of films in comparison with control film.
Micrographs results at
magnification scale
(1000×) showed
significant difference
between the
morphology of polymer
before and after enzyme
entrapment.
Figure 2. Surface morphological analysis of agar-agar film before (a) and
after (b) dextransucrase entrapment at magnifications (10 0 0×).

Entrapped dextransucrase
showed 60% residual activity
after six cycles. Capability of
entrapped enzyme to catalyze
six reactions also suggests it
potential to be used in various
industrial bioprocesses for
continuous product synthesis. Figure 4.Effect of different enzyme : agar-agar ratio on the
immobilization of dextransucrase from Lc. mesenteroides T3
Figure 3. Reusability of entrapped dextransucrase

Considering the economic feasibility, the entrapped DS indicated imperative recycling efficiency up to six reaction cycles. The results of this study revealed that
an easily available and inexpensive matrix, such as agar-agar, could be successfully employed for DS immobilization and OS production.

Acknowledgements
The authors gratefully acknowledge the financial support of Ministry of Science, Education and Technological Development of the Republic of Serbia
under the project TR 31035.
*mmiljkovic@tmf.ac.bg.rs