Characterization of a Novel Thermostable Esterase from Thermus scotoductus SA-01: Evidence of a New Family of Lipolytic Esterases

Plessis, Erika M. du; Berger, Eldie; Stark, Therese; Louw, Maureen E.; Visser, Daniel F.

doi:10.1007/s00284-009-9533-5

Cited by 20 publications

(4 citation statements)

References 31 publications

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“…no. ACS36170; 27.5% similarity according to a Needle (EMBOSS) alignment (http://www.ebi.ac.uk/Tools/psa/) [23]]. A common GXSXG motif that occurs in carboxylesterases and lipases was found in both enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, thermostable lipases have been reported in different Thermus isolates [19], [20] and were recently expressed in thermophilic yeasts [21], [22]. A thermostable esterase from Thermus scotoductus has been reported that was partially biochemically characterized [23]. Finally, two thermostable lipases have been reported from Thermosyntropha lipolytica , an anaerobic, thermophilic, alkali-tolerant bacterium that grows syntrophically with methanogens on lipids [24].…”

Section: Introductionmentioning

confidence: 99%

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The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

et al. 2012

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Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

et al. 2012

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“…While the lipases have the ability to hydrolyse long chain acylglycerols, esterases function by hydrolysing ester substrates with short chain fatty acids. Some examples of bacterial esterases are from Lactobacillus plantarum [20] and Thermus Scotoductus [21].…”

Section: Introductionmentioning

confidence: 99%

The PE-PPE Domain in Mycobacterium Reveals a Serine α/β Hydrolase Fold and Function: An In-Silico Analysis

2011

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The PE and PPE proteins first reported in the genome sequence of Mycobacterium tuberculosis strain H37Rv are now identified in all mycobacterial species. The PE-PPE domain (Pfam ID: PF08237) is a 225 amino acid residue conserved region located towards the C-terminus of some PE and PPE proteins and hypothetical proteins. Our in-silico sequence analysis revealed that this domain is present in all Mycobacteria, some Rhodococcus and Nocardia farcinica genomes. This domain comprises a pentapeptide sequence motif GxSxG/S at the N-terminus and conserved amino acid residues Ser, Asp and His that constitute a catalytic triad characteristic of lipase, esterase and cutinase activity. The fold prediction and comparative modeling of the 3-D structure of the PE-PPE domain revealed a “serine α/β hydrolase” structure with a central β-sheet flanked by α-helices on either side. The structure comprises a lid insertion with a closed structure conformation and has a solvent inaccessible active site. The oxyanion hole that stabilizes the negative charge on the tetrahedral intermediate has been identified. Our findings add to the growing list of serine hydrolases in mycobacterium, which are essential for the maintenance of their impermeable cell wall and virulence. These results provide the directions for the design of experiments to establish the function of PE and PPE proteins.

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“…Sequencing of the two positive clones isolated from the library showed that they both contained the locus TT_C0904 encoding the previously characterized 34 kDa esterase that is responsible for the activity [ 62 ]. There is another example with a library constructed in plasmids from the Thermus scotoductus SA-01 genome, which revealed a new enzyme named Est1 and belonging to a new family of lipolytic enzymes, which shares 76% sequence identity with the putative hydrolase of T. thermophilus HB27 WP_011172791 [ 64 ]. However, we did not isolate this putative hydrolase from the functional screening of our library.…”

Section: Lipolytic Enzymes From Thermus Thermophilus mentioning

confidence: 99%

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes

2015

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Lipolytic enzymes, esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis of ester bonds between alcohols and carboxylic acids, and its formation in organic media. At present, they represent about 20% of commercialized enzymes for industrial use. Lipolytic enzymes from thermophilic microorganisms are preferred for industrial use to their mesophilic counterparts, mainly due to higher thermostability and resistance to several denaturing agents. However, the production at an industrial scale from the native organisms is technically complicated and expensive. The thermophilic bacterium Thermus thermophilus (T. thermophilus) has high levels of lipolytic activity, and its whole genome has been sequenced. One esterase from the T. thermophilus strain HB27 has been widely characterized, both in its native form and in recombinant forms, being expressed in mesophilic microorganisms. Other putative lipases/esterases annotated in the T. thermophilus genome have been explored and will also be reviewed in this paper.

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Characterization of a Novel Thermostable Esterase from Thermus scotoductus SA-01: Evidence of a New Family of Lipolytic Esterases

Cited by 20 publications

References 31 publications

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

The PE-PPE Domain in Mycobacterium Reveals a Serine α/β Hydrolase Fold and Function: An In-Silico Analysis

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes

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