β-d-xylosidase from geobacillus thermoleovorans IT-08: Biochemical
characterization and bioinformatics of the enzyme
a Department of Chemistry, Faculty of
Mathematics and Natural Sciences, Universitas Jember, Jalan Kalimantan 37,
Jember 68121, Indonesia
b Proteomic Laboratory of Institute of Tropical Diseases, Universitas Airlangga, Kampus C Mulyorejo, Surabaya 60115, Indonesia
b Proteomic Laboratory of Institute of Tropical Diseases, Universitas Airlangga, Kampus C Mulyorejo, Surabaya 60115, Indonesia
Abstract
The gene
encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus
thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli;
additionally, characterization and kinetic analysis of GbtXyl43B were carried
out. The gene product was purified to apparent homogeneity showing M r of
72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme
exhibited an optimum temperature and pH of 60 C and 6.0, respectively. In terms
of stability, GbtXyl43B was stable at 60 C at pH 6.0 for 1 h as well as at pH
6-8 at 4 C for 24 h. The enzyme had a catalytic efficiency (k cat/K M)
of 0.0048 ± 0.0010 s-1 mM-1 on
p-nitrophenyl-β-d- xylopyranoside substrate. Thin layer chromatography product
analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose
units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble
xylan was eightfold higher than on soluble xylan. Bioinformatics analysis
showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained
carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel
β-strands) and catalytic module (residues 157 to 604 forming five-bladed
β-propeller fold with predicted catalytic residues to be Asp287 and Glu476).
CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is
proposed as a novel CBM36 subfamily.
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