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

56
views

0
downloads

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

Suggestions

OpenMETU
Core

Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus Tunca, S; Yilmaz, EI; Piret, J; Liras, P; Özcengiz, Gülay (Elsevier BV, 2004-09-01) Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum P-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete...
Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: Effects on cephamycin C production Özcengiz, Gülay; Ünsaldi, Eser; Taşkin, Bilgin; Liras, Paloma; Piret, Jacqueline (Informa UK Limited, 2010-05-01) Abstract In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the w...
PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus Ozturk, ZN; Ögel, Zümrüt Begüm (Springer Science and Business Media LLC, 2000-10-01) The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus p...
Role of the cmcH-ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus Kurt, Aslihan; Alvarez-Alvarez, Ruben; Liras, Paloma; Özcengiz, Gülay (Springer Science and Business Media LLC, 2013-07-01) The effect of the CcaR regulatory protein on expression of the cephamycin C gene cluster is studied. Quantitative reverse transcription PCR (qRT-PCR) expression analysis of the cephamycin biosynthesis genes in the ccaR-disrupted strain, S. clavuligerus ccaR::aph, revealed that in the absence of CcaR, the lat and cmcI genes expression was reduced 2,200-and 1,087-fold compared with the wild type. Expression of pcbAB-pcbC-cefD-cefE-cmcJ-cmcH and blp was 225- to 359-fold lower, while expression of pcbR-pbpA-bla...
Enzymic activity of the K5-type yeast killer toxin and its characterization Izgu, F; Altinbay, D; Sertkaya, A (Informa UK Limited, 2005-11-01) K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120U/mg, and the Michaelis constants K-m, and V-max for lami...

Citation Formats

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, pp. 845–851, 2004, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/34955.