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Molecular cloning and analysis of the Thermus caldophilus ADP-glucose pyrophosphorylase

Paper ID Volume ID Publish Year Pages File Format Full-Text
17891 42704 2007 9 PDF Available
Title
Molecular cloning and analysis of the Thermus caldophilus ADP-glucose pyrophosphorylase
Abstract

Previously, we have purified a highly thermostable ADP-glucose pyrophosphorylase (EC 2.7.7.27; AGPase) from Thermus caldophilus GK-24. In the present paper, we further report the molecular cloning and characterization of the thermostable bacterial AGPase. Using a 0.6-kb DNA probe obtained by polymerase chain reaction (PCR) with a primer set deduced from the N-terminal and internal amino acid sequence, the AGPase gene was cloned. The cloned AGPase gene had a 1245-bp open reading frame that encodes a protein of 414 amino acids. Then, the full-length AGPase gene was further cloned into the pHCE19T(II) vector and was expressed in Escherichia coli DH5α. Western analysis of the recombinant enzyme showed the same immunity as the wild-type enzyme with a molecular mass of ca. 46 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinetic parameters of the recombinant AGPase were basically similar to the wild type. The N-terminal region of T. caldophilus AGPase showed a partial similarity to that of E. coli, and R336 and P295 were identical to those of E. coli AGPase. In addition, sequence comparison revealed that R177 and P235 of T. caldophilus AGPase were aligned with K195 and K247 of E. coli, and K376 with R386, while K195 residue of E. coli was reported to be located in the glucose-1-phosphate (Glc-1-P) substrate binding region.

Keywords
ADP-glucose pyrophosphorylase (ATP:α-glucose-1-phosphate adenylyltransferase; EC 2.7.7.27); Thermus caldophilus GK-24; Molecular cloning; Molecular evolutionary tree
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Molecular cloning and analysis of the Thermus caldophilus ADP-glucose pyrophosphorylase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 41, Issue 4, 3 September 2007, Pages 423–431
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us