NDLI: A pseudo-β-glucosidase in Arabidopsis thaliana: Correction by site-directed mutagenesis, heterologous expression, purification, and characterization

Content Provider	Springer Nature Link
Author	Turan, Y.
Copyright Year	2008
Abstract	Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature β-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZαB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant β-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0–8.5. The purified recombinant β-glucosidase is effectively active on para-/ortho-nitrophenyl-β-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) with K m values of 1.9, 2.1, 0.78 mM and k cat values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-β-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-β-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of β-glucosidases and mutated recombinant enzyme is discussed.
Starting Page	912
Ending Page	919
Page Count	8
File Format	PDF
ISSN	00062979
Journal	Biochemistry (Moscow)
Volume Number	73
Issue Number	8
e-ISSN	16083040
Language	English
Publisher	SP MAIK Nauka/Interperiodica
Publisher Date	2008-08-29
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Arabidopsis thaliana β-glucosidase site-directed mutagenesis heterologous expression Pichia pastoris Biomedicine general Microbiology Bioorganic Chemistry Biochemistry
Content Type	Text
Resource Type	Article
Subject	Biochemistry Biophysics Geriatrics and Gerontology

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