info:eu-repo/semantics/lecture
Effect of precipitants on the kinetic parameters of ?-galactosidase from Aspergillus oryzae and Bacillus circulans immobilized by aggregation and crosslinking
Autor
Guerrero-Siancas, Cecilia Andrea
Vera, Carlos
Illanes, Andrés
Institución
Resumen
-Galactosidases catalyze transglycosylation reactions, allowing
the synthesis of prebiotic transgalactosylated oligosaccharides
(TOS), such as galacto-oligosaccharides (GOS) and lactulose. Immobilization
can dramatically affect the properties of the enzyme,
these alterations serving to the purpose of modulating the
selectivity of the enzyme for a substrate or the formation of
side-products. Different techniques of immobilization by aggregation
and crosslinking of -galactosidases from Aspergillus oryzae
(AOG) and Bacillus circulans (BCG) were evaluated in the hydrolytic
reaction to assess the effect of immobilization in the kinetic parameters
Km and Vmax. Different precipitants (acetone, acetonitrile,
polyethyleneglycol (PEG), ethyl acetate, propanol, isopropanol,
butanol, tert-butanol, pentanol, tert-pentanol, hexanol, heptanol
and octanol) were used for producing cross-linked aggregates
(CLAGs) using glutaraldehyde as cross-linker. All biocatalysts were
evaluated in the reaction of hydrolysis of o-nitrophenyl- -dgalactopyranoside
(o-NPG) comparing their Michaelis constants
(Km) and catalytic efficiencies (Vmax ×Km
−1). Catalytic efficiency of
CLAGs was much higher than the free enzyme. Precipitating agents
had a strong effect on the kinetic parameters of AOG and BCG biocatalysts.
The highest catalytic efficiency was obtained with CLAGs
of AOG precipitated with propanol. Km values increased with the
chain length of the aliphatic alcohol. In the case of BCG, the highest
catalytic efficiencies were obtained with CLAGS precipitated with
PEG and the lowest for those obtained with acetone and acetonitrile.
Precipitant used may also affect the selectivity of CLAGs in
the reactions of transgalactosylation, which opens up interesting
options for increasing yields and productivities of TOS synthesis
with -galactosidases.
Acknowledgement: Work funded by Fondecyt Grant 1160216,
Chile.
http://dx.doi.org/10.1016/j.nbt.2016.06.1099 FONDECYT FONDECYT