Cloning, Sequence, and Expression of the L-(+) Lactate Dehydrogenase of Streptococcus bovis

Wyckoff, H.A.; Chow, John W.; Whitehead, Terence R.; Cotta, Michael A.

doi:10.1007/s002849900197

Cited by 27 publications

(22 citation statements)

References 30 publications

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“…The nucleotide sequence of JB1 Idh was identical to the sequence reported by Wyckoff et al (1997). The nucleotide sequence of TH1 Idh was different from that of JB1 Idh and TH2 Idh at seven points: T345-A, A375 -T, T384 -> A, 0390 A, T564 -A, A630 -> C and A771 -T. However, only one amino acid, Asp220, was replaced by Glu in the amino acids deduced.…”

Section: Cloning Of S Bovis Ldh Genesupporting

confidence: 51%

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

Asanuma

Iwamoto

Hino

1997

J. Gen. Appl. Microbiol.

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Lactate dehydrogenase (LDH) was purified from three strains of Streptococcus bovis, and the gene Idh was cloned and sequenced. The Idh of S. bovis from a goat (TH1) was different from the Idhs of two other strains from cattle (TH2, JB1) in that Asp220 was substituted for Glu. Northern blot analysis revealed that the LDH-mRNA of S. bovis was approximately 1.0 kbp, which was transcribed in a monocistronic fashion. When cells were grown at pH 6.9 in a batch culture, the level of Idh transcript decreased as the growth phase changed; from exponential growth to the cessation of growth. The level of Idh transcript was higher in cells grown at pH 4.5 than at pH 6.9. This observation was consistent with the amounts of LDH in cells and the percentages of lactate produced. These results support the hypothesis that S. bovis regulates LDH synthesis at the transcriptional level probably in response to intracellular pH.

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Section: Cloning Of S Bovis Ldh Genesupporting

confidence: 51%

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

Asanuma

Iwamoto

Hino

1997

J. Gen. Appl. Microbiol.

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“…These results confirm that the TF tag disrupts the combination of monomers into tetramers in LDH. Some reports described the properties of LDH expressed with a tag [20,21]. However, there are few reports comparing the enzymatic properties of expressed and native LDH, except for the report of Johns and Somero [16], who found that the Km values for pyruvate at 20…”

Section: Resultsmentioning

confidence: 99%

“…Many studies on the expression of LDH in E. coli have been reported [20,21], but almost all were on fusion proteins or proteins with relatively large amino acid residues with properties different from the native one in their threedimensional structures and enzyme activities. In this study, we examined the optimum conditions to remove the tag of fused proteins.…”

Section: Introductionmentioning

confidence: 99%

Expression of the Lactate Dehydrogenase Gene fromEptatretus okinoseanusinEscherichia coli

et al. 2011

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Amplified Eptatretus okinoseanus cDNA was digested with NdeI and EcoRI, cloned into pCold trigger factor (TF), and transformed with Escherichia coli strain BL 21 in which a csp A promoter was introduced to inhibit the expression of foreign peptides. Recombinant lactate dehydrogenase (LDH) was obtained in the soluble fraction after sonication of the cells. The protein was digested by HRC 3C protease, thrombin, and factor Xa. The specific activity of TF-tagged protein and tagless protein were 0.646 × 10 6 mIU/mg and 3.56 × 10 6 mIU/mg, respectively. The deletion of the TF tag enhanced the activity compared with the native protein to 13.4 × 10 6 mIU/mg, showing that this expression method is effective for the mass production of the protein to allow further study of the structure of LDH.

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“…Nucleotide comparisons resulted in even lower levels of similarity, which explained the difficulty which I encountered when I used heterologous probes at the beginning of this study. Because no fungal ldh genes had been described previously, I originally tried to make a probe from almost full-length Streptococcus bovis (37), Lactococcus lactis (17), and bovine (12) genes, but I did not detect any specific hybridization with either the cDNA or genomic libraries.…”

Section: Isolation Of Ldh Genesmentioning

confidence: 99%

Isolation and Expression of Lactate Dehydrogenase Genes fromRhizopus oryzae

Skory

2000

Appl Environ Microbiol

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Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD ؉ -dependent L-lactate dehydrogenases (LDH) (EC 1.1.1.27), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts from ldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize that ldhB encodes a second NAD ؉ -dependent LDH that is capable of converting L-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. Both ldhA and ldhB restored fermentative growth to Escherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.Global lactic acid production is estimated to be more than 100,000 tons per year, and approximately 75% of the lactic acid produced is used in the food industry as an acidulant for flavor or as an antimicrobial agent (26). More recent uses for lactic acid have been driven by ecological interest and include production of the nonchlorinated solvent ethyl lactate and the biodegradable plastic polylactic acid. Polylactic acid is a polymer whose properties are similar to those of polyolefins, and it could replace a significant portion of the polyethylene terephthalate-based polymers, which are produced at a rate of approximately 15 million tons per year worldwide (4,14,26). Lactic acid can be synthesized chemically, but such synthesis results in a mixture of D and L isomers. The products of microbiological fermentations depend on the organism used and also may include a mixture the two isomers or individual isomers in a stereospecific form. The desired stereospecificity of the product depends on the intended use; however, L-(ϩ)-lactic acid is the form desired for most applications.In 1936, Lockwood et al. found that in a chemically defined medium, Rhizopus oryzae was able to aerobically convert glucose to large amounts of L-(ϩ)-lactic acid (18). Research on lactic acid production by Rhizopus spp. has continued primarily because of the ease of product purification and the ability of the fungus to utilize both complex carbohydrates and pentose sugars (35,38). Production of lactic acid by Rhizopus cultures is often preferred to bacterial fermentations, because lactobacilli require that the growth medium be supplemented with comp...

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Cloning, Sequence, and Expression of the L-(+) Lactate Dehydrogenase of Streptococcus bovis

Cited by 27 publications

References 30 publications

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

Expression of the Lactate Dehydrogenase Gene fromEptatretus okinoseanusinEscherichia coli

Isolation and Expression of Lactate Dehydrogenase Genes fromRhizopus oryzae

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