Purification and characterization of the malate dehydrogenase from<i>Streptomyces aureofaciens</i>

Mikulášova, D.; Kollárová, Marta; Miginiac‐Maslow, Myroslawa; Decottignies, Paulette; Jacquot, Jean‐Pierre; Kutějová, Eva; Mernik, N.; Egyudová, Ingrid; Musrati, Rabia Ahmed; Horecká, Tatiana

doi:10.1111/j.1574-6968.1998.tb12875.x

Cited by 10 publications

(2 citation statements)

References 35 publications

(32 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sy MDH was stable at a wide range of temperature, being particularly tolerant to high temperatures ( Figure 1C ). Among the mesophilic microorganisms, MDHs from Streptomyces avermitilis , Streptomyces coelicolor , and Nitrosomonas europaea maintain their activity at 50°C ( Mikulášová et al, 1998 ; Ge et al, 2010 ; Deutch, 2013 ), but these MDHs are completely inactivated at 60–70°C ( Mikulášová et al, 1998 ; Ge et al, 2010 ; Deutch, 2013 ). Sy MDH maintains its activity in both oxidative and reductive reactions at 60–70°C ( Figure 1C ).…”

Section: Discussionmentioning

confidence: 99%

“…Since Sy MDH is a heat-stable enzyme ( Figure 1B ), the enzyme activity became the highest at around 50°C, which is higher than the optimal growth temperature in Synechocystis 6803. Besides Synechocystis 6803, microorganisms having the MDHs with the optimal temperature much higher than the optimal growth temperature are S. avermitilis and S. coelicolor , N. europaea ( Mikulášová et al, 1998 ; Ge et al, 2010 ; Deutch, 2013 ). Sy MDH activity was suppressed by copper ( Figure 4 ), as was observed for the MDH from Pseudomonas stutzeri ( Labrou and Clonis, 1997 ).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

et al. 2018

View full text Add to dashboard Cite

Cyanobacteria possess an atypical tricarboxylic acid (TCA) cycle with various bypasses. Previous studies have suggested that a cyclic flow through the TCA cycle is not essential for cyanobacteria under normal growth conditions. The cyanobacterial TCA cycle is, thus, different from that in other bacteria, and the biochemical properties of enzymes in this TCA cycle are less understood. In this study, we reveal the biochemical characteristics of malate dehydrogenase (MDH) from Synechocystis sp. PCC 6803 MDH (SyMDH). The optimal temperature of SyMDH activity was 45–50°C and SyMDH was more thermostable than MDHs from other mesophilic microorganisms. The optimal pH of SyMDH varied with the direction of the reaction: pH 8.0 for the oxidative reaction and pH 6.5 for the reductive reaction. The reductive reaction catalysed by SyMDH was activated by magnesium ions and fumarate, indicating that SyMDH is regulated by a positive feedback mechanism. The Km-value of SyMDH for malate was approximately 210-fold higher than that for oxaloacetate and the Km-value for NAD+ was approximately 19-fold higher than that for NADH. The catalytic efficiency of SyMDH for the reductive reaction, deduced from kcat-values, was also higher than that for the oxidative reaction. These results indicate that SyMDH is more efficient in the reductive reaction in the TCA cycle, and it plays key roles in determining the direction of the TCA cycle in this cyanobacterium.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

et al. 2018

View full text Add to dashboard Cite

show abstract

Metagenome Mining: A Sequence Directed Strategy for the Retrieval of Enzymes for Biocatalysis

et al. 2016

View full text Add to dashboard Cite

Biocatalytic reactions are increasingly being used as a sustainable strategy in organic synthesis and it is recognised that there is need for new enzyme discovery. To establish the utility and versatility of a metagenomics approach, metagenomic DNA extracted from the oral cavity was sequenced and used to create an in silico contig library. This enables individual open reading frames, operons or all the enzymes of a particular family to be identified and then retrieved from the original DNA by PCR. As proof of principle a lactate dehydrogenase, a malate dehydrogenase and transketolases were identified in silico, successfully cloned and assayed. This new enzyme retrieval sequence directed method gives constructive access to metagenomic diversity and importantly improves on the low hit rate experienced when using conventional metagenomic screens.Over the last 30 years enzymes have increasingly been used in commercial chemical processes. [1] Enzyme catalysts have distinct advantages over chemical catalysts such as cost, sustainability, low toxicities and the use of moderate reaction conditions. The greatest advantage however, is the wide range of catalytic activities displayed by naturally occurring enzymes and the stereoselectivities that can be achieved. Drawbacks when using enzymes can include a low organic solvent tolerance, narrow pH working ranges, and low thermostability. However these properties along with enhanced stereoselectivities can be engineered using random or directed evolution techniques. [2] One of the key requirements for this type of engineering is multiple amino acid sequences. Having many enzymes that cat-alyze the same reaction, with variation in their primary sequence is a much more effective starting point for the engineering process than any single example of an enzyme family. One successful method for identifying large numbers of novel enzymes has been to mine fully sequenced and annotated genomes of laboratory cultivable strains. Such sequence directed genome mining lends itself to the retrieval of multiple targets, however a limitation to this method is the number of annotated available species. [3] With only 0.1-5 % of bacteria cultivable in the laboratory, much of the existing diversity of bacteria are inaccessible in this way. [4] At the same time, with the advances in high throughput sequencing, whole or partial genomes of uncultivable bacteria are being made available for study. The nascent field of metagenomics gives insight into the genomes of previously unstudied bacteria and by extension a potential wealth of new biocatalysts for the generation of small molecules and large bioactive molecules such as polyketides and glycopeptides. [5] The use of metagenomics to obtain new enzymes and biocatalysts for industrial applications is steadily growing but still in its infancy. [6] Efforts to capture enzymes from metagenomic samples have relied upon the creation of physical metagenomics libraries in more tractable bacteria. [7] Poor transcription and translation, enzymes with activ...

show abstract

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680

Wang

et al. 2010

Mol Biol Rep

View full text Add to dashboard Cite

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

show abstract

Purification and characterization of the malate dehydrogenase fromStreptomyces aureofaciens

Cited by 10 publications

References 35 publications

Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

Metagenome Mining: A Sequence Directed Strategy for the Retrieval of Enzymes for Biocatalysis

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680

Contact Info

Product

Resources

About