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Cloning and Expression of the glgC Gene from Thermus thermophilus HB27. Purification and Initial Characterization of the Recombinant ADP‐Glucose Pyrophosphorylase
Author(s) -
Watanabe Go,
Meyer Christopher R.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a905-b
Subject(s) - thermus thermophilus , biochemistry , enzyme , recombinant dna , chemistry , thermophile , gene , expression vector , affinity chromatography , microbiology and biotechnology , biology , escherichia coli
The glgC gene product ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes a key step in glucan synthesis and is regulated by allosteric metabolites. Little is known about this enzyme from thermophilic bacteria; engineering of such an ADPG PPase could lead to important industrial applications. The deduced amino acid sequence of the T. thermophilus ADPG PPase has a shorter extreme N‐terminus and a number of amino acid changes compared to other bacterial ADPG PPases. Briefly, the gene was PCR amplified from genomic DNA to introduce unique NcoI and BglII sites and ligated into the pSE420 (Invitrogen) expression vector. The gene was successfully expressed and the activity found to be stable after incubation at 90°C for 10 min in the presence of sulfate, a denaturing condition for other ADPG PPases. The recombinant enzyme was purified to homogeneity with a procedure that included sonication, a heat step, and DEAE, phenyl sepharose, and Blue A (Amicon) chromatography resulting in a ~140‐fold purification (specific activity of 53.6 U/mg and 103 U/mg at 37 and 75°C, respectively) in 34% yield. The T. thermophilus ADPG PPase exhibited a fairly broad pH (6.5–8, most active at pH 7.5) and temperature optimum (60 – 75°C). The S 0.5 values for ATP, G1P, and Mg (pH 7.5, 75°C) were determined to be 0.22, 0.26, and 2.7 mM, respectively. The enzyme was activated ~2.5‐fold by FBP and ~2‐fold by G6P and F6P and significantly inhibited by phosphate, PLP, and ADP, representing a unique regulatory class. Complete kinetic characterization is underway. Supported in part by NSF grant 0448676.