RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment

Hakki, EE
Akkaya, Mahinur
No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be beta alpha beta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus species. A fragment of the gene (ldh), coding for similar to 72% of the lactate dehydrogenase enzyme from Rhizopus oryzae, was amplified using degenerate primers by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The size of the amplified fragment containing beta alpha beta nucleotide binding site, substrate binding site and catalysis/active site is found to be about 700 bp. The reported degenerate PCR primers and the amplification conditions may lead to the cloning of the lactate dehydrogenase gene of R. oryzae, which is an important organism due to its utilization in lactic acid and enzyme productions in industrial scales. (C) 2001 Elsevier Science Inc. All rights reserved.


Kinetics of amino acid production by over-producer mutant microorganisms
Özilgen, Mustafa (Elsevier BV, 1988-2)
Experimental data were modeled based on the literature for the production of nine different amino acids by ten different mutant over-producer microorganisms. Mathematical modeling helped to better understand common aspects of these fermentations, which were not stressed previously by plotting the data only. A Luedeking-Piret type of a model was found to be valid for representing all the amino acid production data. This result indicated the similarity among the amino acid production patterns of all the mutan...
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...
Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads
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A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surf...
Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network
Çalık, Pınar; Oliver, SG; Ozdamar, TH (Elsevier BV, 2003-05-20)
This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes wer...
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...
Citation Formats
E. Hakki and M. Akkaya, “RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment,” ENZYME AND MICROBIAL TECHNOLOGY, pp. 259–264, 2001, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/56582.