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Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described.

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP&#95;004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(½)< 5 min) than the wild-type enzyme (t(½)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(½)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this work, a system for high-level secretion by Saccharomyces cerevisiae of the Thermus thermophilus HB27 putative esterase YP&#95;004875.1 was constructed. The recombinant protein was purified and partially characterised. Its lipolytic activity dropped abruptly when the acyl chain length of the substrate increased from 12 to 18 carbon atoms, and variation of the reaction rate as function of substrate concentration followed Michaelis-Menten kinetics. These results suggested that the enzyme was an esterase. The recombinant enzyme was N-glycosylated and both the glycosylated and non-glycosylated forms showed activity. Compared to the native enzyme, thermal stability (half-life of 4.3h at 85 degrees C) was higher, optimum temperature (40 degrees C) was lower and optimum pH (7.5-8.5) was similar. These characteristics support potential biotechnological applications of the recombinant esterase., (Copyright 2009 Elsevier B.V. All rights reserved.)

Abstract: A membrane-associated esterase (E34Tt) was detected in Thermus thermophilus HB27. The enzyme was purified to homogeneity in a three-step protocol. Detergent (CHAPS) above the CMC was found to be essential to solubilise the enzyme from cell membranes as well as for maintaining activity and stability. By using mass fingerprinting, peptides were found to share identity with the YP_004875 protein, which was annotated as putative esterase in the genome analysis of T. thermophilus HB27, although experimental evidence was lacking. No homology was detected with any known lipase or esterase. However, a comparison with the high-scored sequences from a BLASTp search identified the consensus sequence for lipases/esterases between amino acids 157 and 161 (Gly-Cys-Ser159-Ala-Gly). Further inhibition assays with E600 confirmed that Ser159 was involved in the catalytic mechanism. The monomeric enzyme had a molecular mass of 34kDa and exhibited esterase activity with preference for medium chain-length esters (C10). E34Tt was noticeable for its high thermal stability; the optimal reaction temperature was higher than 80°C and the half-life of thermal inactivation at 85°C was 135min, which makes it even more thermostable than some hyperthermophilic esterases. These properties convert E34Tt into a very attractive enzyme for biotechnological purposes. A theoretical structural model was constructed using as template a prolyl oligopeptidase from Sus scrofa, and a putative catalytic triad (Ser159, Glu255 and His293) with high similarity to the template was identified. [Copyright &y& Elsevier]

Abstract: Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30h at 70°C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60°C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80°C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol. [Copyright &y& Elsevier]