Cloning, expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

Fuciños, Pablo; Atanes, Estrella; López-López, Olalla; Solaroli, Michele; Cerdán, M. Esperanza; González-Siso, María-Isabel; Pastrana, Lorenzo; Rúa, M. Luisa

doi:10.1016/j.procbio.2014.03.006

Cited by 17 publications

(13 citation statements)

References 38 publications

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“…In fact, the three enzymes mentioned in the paragraph above are of thermophilic origin but expressed in mesophilic hosts that grow at 30° ( S. cerevisiae ) or 37°C ( E. coli ). Moreover, a decrease of the optimal temperature was also shown by an esterase from Thermus thermophilus expressed in several yeasts and E. coli (López-López et al, 2010 ; Fuciños et al, 2011 , 2014 ; Rocha et al, 2011 ); the lowest optimum temperature (40°C) was found when S. cerevisiae was the host, while the native T. thermophilus enzyme shows an optimum of temperature at 80°C. The effect of glycosylation and FLAG-tag (due to the S. cerevisiae expression system) on decrease of optimum temperature was discarded (López-López et al, 2010 ).…”

Section: Resultsmentioning

confidence: 95%

Metagenomics of an Alkaline Hot Spring in Galicia (Spain): Microbial Diversity Analysis and Screening for Novel Lipolytic Enzymes

et al. 2015

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A fosmid library was constructed with the metagenomic DNA from the water of the Lobios hot spring (76 • C, pH = 8.2) located in Ourense (Spain). Metagenomic sequencing of the fosmid library allowed the assembly of 9722 contigs ranging in size from 500 to 56,677 bp and spanning ∼18 Mbp. 23,207 ORFs (Open Reading Frames) were predicted from the assembly. Biodiversity was explored by taxonomic classification and it revealed that bacteria were predominant, while the archaea were less abundant. The six most abundant bacterial phyla were Deinococcus-Thermus, Proteobacteria, Firmicutes, Acidobacteria, Aquificae, and Chloroflexi. Within the archaeal superkingdom, the phylum Thaumarchaeota was predominant with the dominant species "Candidatus Caldiarchaeum subterraneum." Functional classification revealed the genes associated to one-carbon metabolism as the most abundant. Both taxonomic and functional classifications showed a mixture of different microbial metabolic patterns: aerobic and anaerobic, chemoorganotrophic and chemolithotrophic, autotrophic and heterotrophic. Remarkably, the presence of genes encoding enzymes with potential biotechnological interest, such as xylanases, galactosidases, proteases, and lipases, was also revealed in the metagenomic library. Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. Six genes conferring lipolytic activity were identified and one was cloned and characterized. This gene was named LOB4Est and it was expressed in a yeast mesophilic host. LOB4Est codes for a novel esterase of family VIII, with sequence similarity to β-lactamases, but with unusual wide substrate specificity. When the enzyme was purified from the mesophilic host it showed half-life of 1 h and 43 min at 50 • C, and maximal activity at 40 • C and pH 7.5 with p-nitrophenyl-laurate as substrate. Interestingly, the enzyme retained more than 80% of maximal activity in a broad range of pH from 6.5 to 8.

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Section: Resultsmentioning

confidence: 95%

Metagenomics of an Alkaline Hot Spring in Galicia (Spain): Microbial Diversity Analysis and Screening for Novel Lipolytic Enzymes

et al. 2015

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“…Some thermostable enzymes are still recovered by isolation from thermophilic microorganisms (Shi et al, 2013 ; Fuciños et al, 2014 ; Sen et al, 2016 ), however metagenomics has opened a new important field in the discovery of novel biocatalysts and has been revealed as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. There are two different ways of screening a metagenome in search of thermozymes: a sequence-based approach and a function-based approach (Figure 2 ).…”

Section: Metagenome-derived Thermozymesmentioning

confidence: 99%

“…Therefore, screening for novel biocatalysts from extremophiles has become a very important field. In the last few years, novel thermostable polymerases (Moser et al, 2012 ; Schoenfeld et al, 2013 ), beta-galactosidases (Wang et al, 2014 ), esterases (Fuciños et al, 2014 ), and xylanases (Shi et al, 2013 ), among others, have been described and characterized, opening a new horizon in biotechnology.…”

Section: Introductionmentioning

confidence: 99%

Metagenomics of Thermophiles with a Focus on Discovery of Novel Thermozymes

2016

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Microbial populations living in environments with temperatures above 50°C (thermophiles) have been widely studied, increasing our knowledge in the composition and function of these ecological communities. Since these populations express a broad number of heat-resistant enzymes (thermozymes), they also represent an important source for novel biocatalysts that can be potentially used in industrial processes. The integrated study of the whole-community DNA from an environment, known as metagenomics, coupled with the development of next generation sequencing (NGS) technologies, has allowed the generation of large amounts of data from thermophiles. In this review, we summarize the main approaches commonly utilized for assessing the taxonomic and functional diversity of thermophiles through metagenomics, including several bioinformatics tools and some metagenome-derived methods to isolate their thermozymes.

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“…The need for stable enzymes that tolerate alkaline conditions also justifies the screening and characterization of extremozymes (Box 1). Thermostable lipases of archaeal origin that prefer slightly alkaline conditions were recently identified from Sulfolobus acidophilus and Pyrobaculum sp., while lipolytic thermozymes that are optimally active at acidic conditions were exclusively isolated from Thermotoga maritima and Thermus thermophilus (Table 1) [35][36][37][38]. Especially the esterases from S. acidophilus and Pyrobaculum sp.…”

Section: Lipolytic Enzymesmentioning

confidence: 99%