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Title 

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

Authors 

Yong Sam KimH SohnU H JinS J SuhSang Chul LeeJae Heung JeonDae Sil LeeC H KimJeong Heon Ko

Publisher 

Elsevier

Issue Date 

2007

Citation 

Enzyme and Microbial Technology, vol. 41, no. 4, pp. 423-431

Keywords 

ADP-glucose pyrophosphorylase (ATP:α-glucose-1-phosphate adenylyltransferaseEC 2.7.7.27)molecular cloningmolecular evolutionary treethermus caldophilus GK-24

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.

ISSN 

0141-0229

Link 

http://dx.doi.org/10.1016/j.enzmictec.2007.03.010

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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