Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization

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dc.contributor.authorÖzkan, M
dc.contributor.authorYilmaz, EI
dc.contributor.authorLynd, LR
dc.contributor.authorÖzcengiz, G
dc.date.accessioned2024-04-24T17:12:00Z
dc.date.available2024-04-24T17:12:00Z
dc.date.issued2004
dc.departmentDicle Üniversitesien_US
dc.description.abstractThe 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.en_US
dc.identifier.doi10.1139/W04-071
dc.identifier.endpage851en_US
dc.identifier.issn0008-4166
dc.identifier.issn1480-3275
dc.identifier.issue10en_US
dc.identifier.pmid15644899
dc.identifier.scopus2-s2.0-14344260653
dc.identifier.scopusqualityQ3
dc.identifier.startpage845en_US
dc.identifier.urihttps://doi.org/10.1139/W04-071
dc.identifier.urihttps://hdl.handle.net/11468/17814
dc.identifier.volume50en_US
dc.identifier.wosWOS:000225904800009
dc.identifier.wosqualityQ3
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakScopus
dc.indekslendigikaynakPubMed
dc.language.isoenen_US
dc.publisherCanadian Science Publishing, Nrc Research Pressen_US
dc.relation.ispartofCanadian Journal of Microbiology
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanıen_US
dc.rightsinfo:eu-repo/semantics/closedAccessen_US
dc.subjectL-Lactate Dehydrogenase Purificationen_US
dc.subjectThermophilic Bacteriaen_US
dc.titleCloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterizationen_US
dc.titleCloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization
dc.typeArticleen_US

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