Abstract
Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S(3)N(10) peptide known to be completely resistant to E. coli endopeptidase. The CBD-GAD expressed in E. coli was successfully immobilized on Avicel, a crystalline cellulose, with binding capacity of 33 ± 2 nmol(CBD-GAD)/g(Avicel) and the immobilized enzymes retained 60% of their initial activities after 10 uses. The results of this report provide a feasible alternative to produce GABA using immobilized GAD through fusion to CBD.
Keywords:
GAD; cellulose-binding domain; fusion protein; immobilization.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cellulase / chemistry
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Cellulase / metabolism
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Cellulose / chemistry
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Cellulose / metabolism*
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Endopeptidases / metabolism
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Enzymes, Immobilized / chemistry
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Enzymes, Immobilized / metabolism
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Escherichia coli / enzymology
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Escherichia coli / metabolism
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Glutamate Decarboxylase / chemistry
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Glutamate Decarboxylase / genetics
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Glutamate Decarboxylase / metabolism*
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Peptides / chemistry
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Peptides / genetics
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Peptides / metabolism
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Protein Binding
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Proteolysis
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Trichoderma / enzymology
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gamma-Aminobutyric Acid / metabolism
Substances
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Enzymes, Immobilized
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Peptides
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Recombinant Fusion Proteins
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gamma-Aminobutyric Acid
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Cellulose
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endoglucanase 2
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Cellulase
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Endopeptidases
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Glutamate Decarboxylase