(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Firmicutes: NE > Clostridia: NE > Thermoanaerobacterales: NE > Thermoanaerobacteraceae: NE > Thermoanaerobacter: NE > Thermoanaerobacter pseudethanolicus: NE > Thermoanaerobacter pseudethanolicus ATCC 33223: NE
ACPH_Peptidase_S9 : thep3-b0kcf0Thermoanaerobacter pseudethanolicus (Clostridium thermohydrosulfuricum), T. ethanolicus, T. brockii (T. finnii), T. mathranii, T. italicus, T. sp., esterase. Homoserine_transacetylase : thep3-b0k721Thermoanaerobacter ethanolicus ATCC 33223 homoserine o-acetyltransferase (EC 2.3.1.31). Prolyl_oligopeptidase_S9 : thep3-b0k8d6Thermoanaerobacter pseudethanolicus (Clostridium thermohydrosulfuricum), T. ethanolicus, T. brockii (T. finnii), T. sp., peptidase s9, prolyl oligopeptidase active site region
Warning: This entry is a compilation of different species or line or strain with more than 90% amino acid identity. You can retrieve all strain data
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) Thermoanaerobacter pseudethanolicus ATCC 33223: N, E.
Thermoanaerobacter thermohydrosulfuricus: N, E.
Thermoanaerobacter brockii subsp. finnii Ako-1: N, E.
Thermoanaerobacter sp. X514: N, E.
Thermoanaerobacter italicus: N, E.
Thermoanaerobacter italicus Ab9: N, E.
Thermoanaerobacter mathranii subsp. mathranii str. A3: N, E.
Thermoanaerobacter ethanolicus CCSD1: N, E.
Thermoanaerobacter sp. X561: N, E.
Thermoanaerobacter sp. X513: N, E.
Thermoanaerobacter ethanolicus JW 200: N, E.
unclassified Thermoanaerobacter: N, E.
Thermoanaerobacter: N, E.
Molecular evidence
Database
No mutation 3 structures: 7Q4H, 7Q4J, 8B9S No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MQKAVEITYNGKTLRGMMHLPDDVKGKVPMVIMFHGFTGNKVESHFIFVK MSRALEKVGIGSVRFDFYGSGESDGDFSEMTFSSELEDARQILKFVKEQP TTDPERIGLLGLSMGGAIAGIVAREYKDEIKALVLWAPAFNMPELIMHES VKQYGAIMEQLGFVDIGGHKLSKDFVEDISKLNIFELSKGYDKKVLIVHG TNDEAVEYKVSDRILKEVYGDNATRVTIENADHTFKSLEWEKKAIEESVE FFKKELLKG
Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.
Title: Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology Pournejati R, Karbalaei-Heidari HR, Budisa N Ref: Protein Expr Purif, 101:84, 2014 : PubMed
Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration.
Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90 degrees C and between pH 6.5 and 10; the half-life of the enzymes at 75 degrees C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53-2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.
Thermoanaerobacter thermohydrosulfuricus; Thermoanaerobacter ethanolicus TTL lipase LipTth
