Gene expression and characterization of thermostable glutamate decarboxylase from Pyrococcus furiosus

Lee, Eon-Seok; Kim, Han-Woo; Kim, Dong-Eun; Kim, Yeon-Hee; Nam, Soo Woo; Kim, Byung-Woo; Jeon, Sung-Jong

doi:10.1007/s12257-012-0581-5

Cited by 8 publications

(3 citation statements)

References 26 publications

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“…GABA can be used as a carbon or energy source in eukaryotes and bacteria (Kumar and Punekar, 1997; Shelp et al ., 1999), but the absence of related transporters and catabolism pathways for this substrate in Halosegnis genomes, suggest it must play a role in the cell. In some bacteria, GABA is involved in acid or stress resistance (Coleman et al ., 2001; Cao et al ., 2013) and several studies in archaea and bacteria for its potential in the food, nutraceuticals and pharmaceutical fields (Park and Oh, 2007; Siragusa et al ., 2007; Kim et al ., 2009; Lee et al ., 2013) have been carried out. The probable synthesis of GABA by the new isolates of the genus Halosegnis indicates its potential in biotechnological applications.…”

Section: Resultsmentioning

confidence: 99%

Culturomics‐based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis

Durán-Viseras

Andrei

Vera-Gargallo

et al. 2020

Environmental Microbiology

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The development of culture-independent techniques has revolutionized our understanding of microbial ecology, especially through the illustration of the vast gap between the environmentally abundant microbial diversity and that accessible through cultivation. However, culture-based approaches are not only crucial for understanding the evolutionary, metabolic and ecological milieu of microbial diversity but also for the development of novel biotechnological applications. In this study, we used a culturomics-based approach in order to isolate novel microbial taxa from hypersaline environments (i.e. Isla Cristina and Isla Bacuta salterns in Huelva, Spain). We managed to obtain axenic cultures of four haloarchaeal strains that belong to a new haloarchaeal genus and to obtain their genomic sequences. The phylogenomic and phylogenetic analyses (together with AAI, ANI and digital DDH indices) showed that the isolates constitute two new species, for which we propose the names Halosegnis longus sp. nov. and Halosegnis rubeus sp. nov. The genomic-based metabolic reconstructions indicated that members of this new haloarchaeal genus have photoheterotrophic aerobic lifestyle with a typical salt-in signature. 16S rRNA gene sequence reads abundance profiles and genomic recruitment analyses revealed that the Halosegnis genus has a worldwide geographical distribution, reaching high abundance (up to 8%) in habitats with intermediate salinities.

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Section: Resultsmentioning

confidence: 99%

Culturomics‐based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis

Durán-Viseras

Andrei

Vera-Gargallo

et al. 2020

Environmental Microbiology

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“…These processes result in contamination of the recombinant protein by various cellular components. Removing them requires costly down-stream processing [ 15 – 17 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C

Jiang

et al. 2017

Microb Cell Fact

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BackgroundOur laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins.ResultsWhen B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process.ConclusionsThis is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

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“…However, almost all carboxylic ester hydrolases are mesophilic enzymes not suitable for harsh industrial processes, such as organic solvents, high temperature, and alkali [3]. Enzymes from hyperthermophiles are promising candidates because of their high intrinsic thermal and chemical stability [4,5]. Therefore, production of the enzymes from thermophiles becomes a hot topic.…”

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of a Novel Thermo-Alkalistable Lipase from Hyperthermophilic Bacterium Thermotoga maritima

Tian

Chen

et al. 2015

Appl Biochem Biotechnol

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A gene coding for lipase (Tm1350) from the hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned and overexpressed by Escherichia coli. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent Km and Vmax values for p-nitrophenyl butyrate were 8 mM and 333 U/mg, respectively. The enzyme displayed optimal activity at pH 7.5 and 70 °C, maintained 66 % of the original activity after 8 h of incubation, and its half-lives at pHs 9 and 10 were 8 and 1 h. The activity of Tm1350 was stimulated up to 131 or 151 % of the original activity by incubating with 4 M urea or 20 % (v/v) methanol, and 90.1 or 70.2 % of the activity was maintained after 8 h incubation of the enzyme in 20 or 75 % (v/v) of the methanol, showing potential for biodiesel production. The activity of the enzyme without cysteine residue was stimulated up to 618 and 550 % of the original activity by incubating with dithiothreitol (DTT) and reduced glutathione (GSH) at a concentration of 1 mM. However, the circular dichroism spectra of the enzyme have no obvious change after DTT treatment. It is speculated that DTT interacts with potential residues in some key active sites without influence of structure.

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Gene expression and characterization of thermostable glutamate decarboxylase from Pyrococcus furiosus

Cited by 8 publications

References 26 publications

Culturomics‐based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis

Culturomics‐based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis

Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C

Expression and Characterization of a Novel Thermo-Alkalistable Lipase from Hyperthermophilic Bacterium Thermotoga maritima

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