Purification of recombinant mandelate racemase: Improved catalytic activity

Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg²⁺-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate and has been studied extensively as a model for understanding how enzymes catalyze the deprotonation of carbon acid substrates with relatively high pK_a values. Purification of recombinant MR as a fusion protein with an N-terminal hexahistidine tag using immobilized-nickel ion affinity chromatography and elution with a linear gradient of EDTA revealed three enzyme species (mrI, mrII, and mrIII). While mrIII was catalytically inactive, both mrI and mrII catalyzed the racemization of (S)-mandelate with turnover numbers (k_cat) of 190 ± 22 and 940 ± 24 s^?1, respectively. Circular dichroism analysis suggested that mrIII was a partially unfolded or misfolded form of the enzyme. Replacement of the N-terminal hexahistidine tag by a StrepII-tag appeared to ameliorate the folding problem yielding a single enzyme species with a turnover number of 1124 ± 43 s^?1. The MR fusion protein bearing an N-terminal StrepII-tag and a C-terminal decahistidine tag also exhibited reduced turnover (k_cat = 472 ± 37 s^?1). These results highlight a potential problem that may be encountered when producing fusion enzymes bearing a polyhistidine tag: soluble, active enzyme may be obtained but care must be taken to ensure that it is free of minor misfolded forms that can alter the apparent activity of the enzyme.     

Nanoparticle targeting to neurons in a rat hippocampal slice culture model

We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP₉GGH₆ (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.     

“Use of acidophilic bacteria of the genus Acidithiobacillus to biosynthesize CdS fluorescent nanoparticles (quantum dots) with high tolerance to acidic pH”

The use of bacterial cells to produce fluorescent semiconductor nanoparticles (quantum dots, QDs) represents a green alternative with promising economic potential. In the present work, we report for the first time the biosynthesis of CdS QDs by acidophilic bacteria of the Acidithiobacillus genus. CdS QDs were obtained by exposing A. ferrooxidans, A. thiooxidans and A. caldus cells to sublethal Cd²⁺ concentrations in the presence of cysteine and glutathione. The fluorescence of cadmium-exposed cells moves from green to red with incubation time, a characteristic property of QDs associated with nanocrystals growth. Biosynthesized nanoparticles (NPs) display an absorption peak at 360 nm and a broad emission spectra between 450 and 650 nm when excited at 370 nm, both characteristic of CdS QDs. Average sizes of 6 and 10 nm were determined for green and red NPs, respectively. The importance of cysteine and glutathione on QDs biosynthesis in Acidithiobacillus was related with the generation of H₂S. Interestingly, QDs produced by acidophilic bacteria display high tolerance to acidic pH. Absorbance and fluorescence properties of QDs was not affected at pH 2.0, a condition that totally inhibits the fluorescence of QDs produced chemically or biosynthesized by mesophilic bacteria (stable until pH 4.5–5.0). Results presented here constitute the first report of the generation of QDs with improved properties by using extremophile microorganisms.     

Synthesis of highly luminescent and biocompatible CdTe/CdS/ZnS quantum dots using microwave irradiation: a comparative study of different ligands

We compared the effects of several ligands frequently used in aqueous synthesis, including L‐cysteine, L‐cysteine hydrochloride, N‐acetyl‐L‐cysteine (NAC), glutathione and 3‐mercaptopropionic acid, for microwave synthesis of CdTe quantum dots (QDs) in a sealed vessel with varied temperatures and times, and then developed a rapid microwave‐assisted protocol for preparing highly luminescent, photostable and biocompatible CdTe/CdS/ZnS core–multishell QDs. The effects of molecular structures of these ligands on QD synthesis under high temperatures were explored. Among these ligands, NAC was found to be the optimal ligand in terms of the optical properties of resultant QDs and reaction conditions. The emission wavelength of NAC‐capped CdTe QDs could reach 700 nm in 5 min by controlling the reaction temperature, and the resultant CdTe/CdS/ZnS core–multishell QDs could achieve the highest quantum yields up to 74% with robust photostability. In addition, the effects of temperature, growth time and shell–precursor ratio on shell growth were examined. Finally, cell culturing indicated the low cytotoxicity of CdTe/CdS/ZnS core–multishell QDs as compared to CdTe and CdTe/CdS QDs, suggesting their high potential for applications in biomedical imaging and diagnostics. Copyright © 2014 John Wiley & Sons, Ltd.     

Soluble expression and purification of a functional harpin protein in Escherichia coli

The use of harpins in practical agricultural applications may enhance plant growth and induce disease resistance. However, few investigations focused on the optimal expression and purification of harpin. In this work, harpin protein fused with a thioredoxin tag and a hexahistidine tag was expressed in Escherichia coli BL21 (DE3) cells as a soluble form under the induction of 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside. The purity of the recombinant harpin was greater than 90% after one-step nickel-nitrilotriacetic acid affinity chromatography. The yield of purified TRX-harpin protein reached 17.1 mg per 100 mL of cell culture. TRX-harpin is thermostable and could trigger the hypersensitive response effect in tobacco, with an efficient dose as low as 30 μg/mL. The root lengths of TRX-harpin treated tobacco and wheat plants were nearly 1.6-fold and 1.8-fold longer, respectively, than plants treated with the empty vector preparation. Thus, using a N-terminal TRX-tagged fusion is an economic way to produce bioactive harpin.     

Crystallographic and spectroscopic characterizations of Sulfolobus solfataricus TrxA1 provide insights into the determinants of thioredoxin fold stability

Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe pathologies and for their stable scaffold. Here we report a combined structural and spectroscopic characterization of a Trx isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsTrxA1). Thermal denaturation unveils that SsTrxA1 is endowed with a remarkable stability in the explored temperature range 50–105 °C. The structure of the oxidized form of SsTrxA1 determined at 1.9 Å resolution presents a number of peculiar features. Although the protein was crystallized in a slightly acid medium (pH 6.5) as many as ten intramolecular/intermolecular carboxyl–carboxylate interactions involving glutamic and aspartic acid side chains are found in three independent SsTrxA1 molecules present in the asymmetric unit. Surprisingly for a hyperthermostable protein, the structure of SsTrxA1 is characterized by the presence (a) of a very limited number of intramolecular salt bridges and (b) of a cavity nearby Cys52, a residue that is frequently a phenylananine in other members of the family. Chemical denaturation investigations carried out on SsTrxA1 and SsTrxA2 show that both proteins present a significant stability against guanidine hydrochloride, thus indicating that ionic interactions play a minor role in their stabilization. Compared to Trxs from mesophilic sources, SsTrxA1 displays a longer α-helix 1 and a shorter loop connecting this α-helix with β-strand 2. As these features are shared with Trxs isolated from thermophilic sources, the shortening of this loop may be a general strategy adopted to stabilize this fold. This feature may be exploited for the design of hyperthermostable Trx scaffolds.     

Microbial Uptake,Toxicity, and Fate of Biofabricated ZnS:Mn Nanocrystals

Despite their importance in nano-environmental health and safety, interactions between engineered nanomaterials and microbial life remain poorly characterized. Here, we used the model organism E. coli to study the penetration requirements, subcellular localization, induction of stress responses, and long-term fate of luminescent Mn-doped ZnS nanocrystals fabricated under “green” processing conditions with a minimized ZnS-binding protein. We find that such protein-coated quantum dots (QDs) are unable to penetrate the envelope of unmodified E. coli but readily translocate to the cytoplasm of cells that have been made competent by chemical treatment. The process is dose-dependent and reminiscent of bacterial transformation. Cells that have internalized up to 0.5 μg/mL of nanocrystals do not experience a significant activation of the unfolded protein or SOS responses but undergo oxidative stress when exposed to high QD doses (2.5 μg/mL). Finally, although they are stable in quiescent cells over temperatures ranging from 4 to 42°C, internalized QDs are rapidly diluted by cell division in a process that does not involve TolC-dependent efflux. Taken together, our results suggest that biomimetic QDs based on low toxicity inorganic cores capped by a protein shell are unlikely to cause significant damage to the microbial ecosystem.     

Oligohis‐tags: mechanisms of binding to Ni2+‐NTA surfaces

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni²⁺ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C‐ or N‐terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His‐tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His‐tags to Ni²⁺‐NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid‐phase peptide synthesis (SPPS). Binding to Ni²⁺‐NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K_D) of 14 ± 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Zinc-mediated Reversible Multimerization of Hsp31 Enhances the Activity of Holding Chaperone

Hsp31 protein, belonging to the DJ-1/ThiJ/PfpI superfamily, increases the survival of Escherichia coli under various stresses. While it was reported as a holding chaperone, Hsp31 was also shown to exhibit the glyoxalase III activity in subsequent study. Here, we describe our finding that Hsp31 undergoes a Zn^+ 2-mediated multimerization (HMW_Zinc), resulting in an enhanced chaperone activity. Furthermore, it was shown that the formation of HMW_Zinc is reversible such that the oligomer dissociates into the native dimer by EDTA incubation. We attempted to determine the structural change involving the transition between the native dimer and HMW_Zinc by adding Ni^+ 2, which is Zn^+ 2-mimetic, producing a potential intermediate structure. An analysis of this intermediate revealed a structure with hydrophobic interior exposed, due to an unfolding of the N-terminal loop and the C-terminal β-to-α region. A treatment with hydrogen peroxide accelerated HMW_Zinc formation, so that the Hsp31^C185E mutant rendered the formation of HMW_Zinc even at 45 °C. However, the presence of Zn^+ 2 in the catalytic site antagonizes the oxidation of C185, implying a negative role. Our results suggest an unprecedented mechanism of the enhancing chaperone activity by Hsp31, in which the reversible formation of HMW_Zinc occurs in the presence of heat and Zn^+ 2 ion.     

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (K_d = 0.52 nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC₅₀ = 0.098 μM, NCI-N87 IC₅₀ = 0.066 μM) and also displayed high oral bioavailability in mice (F = 44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition = 136%).     

A robust and fast bacteria counting method using CdSe/ZnS core/shell quantum dots as labels

A rapid, sensitive fluorescence measurement method for detecting the bacterial count using CdSe/ZnS as a fluorescence marker was described. High-quality CdSe/ZnS nanocrystals were synthesized and successfully conjugated with bacteria. The fluorescence intensity was proportional to bacterial count in the range of 10²–10⁸ CFU/mL and the low detection limit was 10² CFU/mL.     

Hsp90 inhibition: Elimination of shock and stress

The 90 kDa heat shock proteins (Hsp90) represent a class of molecular chaperones responsible for the maturation and stabilization of many oncogenic proteins. Disrupting the ability of ATP to bind and facilitate the operation of Hsp90 has emerged as a promising approach toward cancer chemotherapeutic development. While numerous Hsp90 inhibitory scaffolds have been identified, progress through the clinic has revealed many obstacles that should be addressed in future analogue development. Recent reports of the complications, pitfalls, and downstream effects associated with Hsp90 inhibition are discussed herein, in hopes of providing a reference that can be used to guide the future design of Hsp90 inhibitory scaffolds.     

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from heat shock protein 70 of rice

Hsp70(241–258), an octadecapeptide derived from the heat shock protein 70 (Hsp70) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains four lysine, two arginine, and two histidine residues. The antimicrobial activity of Hsp70(241–258) against Porphyromonas gingivalis, a periodontal pathogen, and Candida albicans, an opportunistic fungal pathogen, was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentrations of Hsp70(241–258) against P. gingivalis and C. albicans cells were 63 μM and 70 μM, respectively. Hsp70(241–258) had little or no hemolytic activity even at 1 mM, and showed negligible cytotoxicity up to 300 μM. The degrees of calcein leakage from large unilamellar vesicles, which mimic the membranes of Gram-negative bacteria, and 3,3′-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Hsp70(241–258) increased in a concentration-dependent manner. When Hsp70(241–258) was added to calcein-acetoxymethyl ester-loaded C. albicans cells, calcein release from the cells increased in a concentration-dependent manner. Flow cytometric analysis also showed that the percentages of C. albicans cells stained with propidium iodide, a DNA-intercalating dye, increased as the concentration of Hsp70(241–258) added was increased. Therefore, Hsp70(241–258) appears to exhibit antimicrobial activity against P. gingivalis and C. albicans through membrane disruption. These results suggest that Hsp70(241–258) could be useful as a safe and potent AMP against P. gingivalis and C. albicans in many fields of health care, especially in the control of oral infections.     

Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae

The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37 °C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20 °C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37 °C and 20 °C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH.     

Integrity of N‐ and C‐termini is important for E. coli Hsp31 chaperone activity

Hsp31 is a stress‐inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high‐affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20‐residue‐long hexahistidine tag to the N‐termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C‐termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging.     

Differential properties of native and tagged or untagged recombinant glucose isomerases of Streptomyces sp. SK and possible implication of the glycosylation

A comparative study was performed to investigate the biochemical properties of the native glucose isomerase produced by Streptomyces sp. SK (SKGI) and its two recombinant forms, the common recombinant (r-SKGI) and the tagged (His6-SKGI) isomerases. The findings revealed that the three glucose isomerases displayed different behaviors in particular in terms of thermoactivity, stability at high temperature and specific activity. The thermoactivity/thermostablity and specific activity of r-SKGI were lower than those of native SKGI. SDS-PAGE analyses revealed that native and r-SKGI showed different molecular weights. This could be attributed to the presence of 8% O-glycosylation in the SKGI monomers, which entailed a potential attachment of sugar residues having a total mass of about 3.44 kDa on Thr at positions 6 and/or 30. The results also demonstrated that the His-tagged SKGI was less thermoactive/thermostable than SKGI and r-SKGI. Electrophoretic analysis showed that the His tag significantly affected the dimerization of SKGI. A 3D model of His6-SKGI was constructed. The results showed that the Histidine attachment was located at the dimerization interface and that, unlike the rest of this interface, it was hydrophilic in nature, which presumably hamper the dimerization process and led to the decrease in thermoactivity/thermostablity and catalytic efficiency.     

Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu²⁺/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l-p-hydroxyphenylhydantoin (d,l-HPH) to N-carbamoyl-d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation.     

Mass spectral characterization of organophosphate-labeled,tyrosine-containing peptides: Characteristic mass fragments and a new binding motif for organophosphates

We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8–8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.     

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta)

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34 °C and 36 °C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36 °C, 3 h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.     

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

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His₆ was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His₆ preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85 °C), suggesting that E34Tt-His₆ has potential for industrial applications.