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Application of xylanolytic enzymes in alkyl-b-xyloside
synthesis and purification of
b-xylosidase from Sclerotinia
sclerotiorum
Islem Abid1,
Mohamed Gargouri1*, Issam Smaali1, Ferid Limam2,
Thierry Maugard3, Marie Dominique Legoy3 and
Nejib Marzouki3
1
National Institute of Applied Science and Technology (INSAT),
Biological Engineering Unit, Tunis, Tunisia.
mohamed.gargouri@insat.rnu.tn
2
National Institute of Scientific and Technical Research(INRST), Borj
Cedria, Tunisia.
3
Laboratoire de Biotechnologie et de Chimie Bio-organique, Université
de La Rochelle, France.
Abstract:
b-Xylosidase
and endo-xylanase belong to an enzyme battery that phytopathogenic
fungi use for the degradation of the cell walls. An enzyme preparation
from Sclerotinia sclerotiorum was applied in synthesis of
biosurfactants: hexyl-b-xyloside
and hexyl-b-xylobioside
from xylan and hexanol. Also, in order to characterize the enzyme
activity, the
b-xylosidase
obtained in the culture filtrate was purified by the successive use of
anion exchange chromatography DEAE-sepharose, anion exchange HPLC
TSK-DEAE, gel filtration on TSK-200 SW HPLC followed by preparative
native-PAGE.
b-Xylosidase
seems to be a monomeric protein with 70 kDa estimated by gel
filtration and 72 kDa determined by SDS-PAGE. With pNPX as substrate,
b-xylosidase
has Michaelis-Menten kinetics: KM = 1.61 mM and VM
= 0.14 µmol min-1 mg protein. The enzyme showed an optimal
activity at 60 °C and pH 4. It also showed stability over a wide pH
range (from 2 to 9) and retained up to 50 % of its activity at 50 °C.
The enzyme activity was further characterized by testing several
divalent ions and reagents.
Key words:
Alkyl-xyloside, endoxylanase, Sclerotinia sclerotiorum,
synthesis reaction, xylan, b-xylosidase.
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