Show/Hide Menu
Hide/Show Apps
anonymousUser
Logout
Türkçe
Türkçe
Search
Search
Login
Login
OpenMETU
OpenMETU
About
About
Open Science Policy
Open Science Policy
Communities & Collections
Communities & Collections
Help
Help
Frequently Asked Questions
Frequently Asked Questions
Videos
Videos
Thesis submission
Thesis submission
Publication submission with DOI
Publication submission with DOI
Publication submission
Publication submission
Contact us
Contact us
Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization
Date
2004-10-01
Author
Ozkan, M
Yilmaz, EI
Lynd, LR
Özcengiz, Gülay
Metadata
Show full item record
This work is licensed under a
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License
.
Item Usage Stats
11
views
0
downloads
Cite This
The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap 11 phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had K-m and V-max values of 7.3 mmol/L and 87 mumol/min, respectively, and in the presence of FDP, a 24-fold decrease in K-m and a 5.7-fold increase in V-max were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas K-m and V-max values were 59.5 mmol/L and 52 mumol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65degreesC for 5 min.
Subject Keywords
Immunology
,
Applied Microbiology and Biotechnology
,
Genetics
,
Molecular Biology
,
Microbiology
,
General Medicine
URI
https://hdl.handle.net/11511/34955
Journal
CANADIAN JOURNAL OF MICROBIOLOGY
DOI
https://doi.org/10.1139/w04-071
Collections
Department of Biology, Article
Citation Formats
IEEE
ACM
APA
CHICAGO
MLA
BibTeX
M. Ozkan, E. Yilmaz, L. Lynd, and G. Özcengiz, “Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization,”
CANADIAN JOURNAL OF MICROBIOLOGY
, vol. 50, no. 10, pp. 845–851, 2004, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/34955.