Expression, purification, refolding and characterization of a putative lysophospholipase from Pyrococcus furiosus: retention of structure and lipase/esterase activity in the presence of water-miscible organic solvents at high temperatures

A putative lysophospholipase (PF0480) encoded by the Pyrococcus furiosus genome has previously been cloned and expressed in Escherichia coli. Studies involving crude extracts established the enzyme to be an esterase; however, owing presumably to its tendency to precipitate into inclusion bodies, purification and characterization have thus far not been reported. Here, we report the overexpression and successful recovery and refolding of the enzyme from inclusion bodies. Dynamic light scattering suggests that the enzyme is a dimer, or trimer, in aqueous solution. Circular dichroism and fluorescence spectroscopy show, respectively, that it has mixed beta/alpha structure and well-buried tryptophan residues. Conformational changes are negligible over the temperature range of 30–80 °C, and over the concentration range of 0–50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. The enzyme is confirmed to be an esterase (hydrolyzing p-NP-acetate and p-NP-butyrate) and also shown to be a lipase (hydrolyzing p-NP-palmitate), with lipolytic activity being overall about 18- to 20-fold lower than esterase activity. Against p-NP-palmitate the enzyme displays optimally activity at pH 7.0 and 70 °C. Remarkably, over 50% activity is retained at 70 °C in the presence of 25% acetonitrile. The high organic solvent stability and thermal stability suggest that this enzyme may have useful biodiesel-related applications, or applications in the pharmaceutical industry, once yields are optimized.     

Translocation of metals in pea plants grown on various amendment of electroplating industrial sludge

A pot-culture experiment was conducted to observe the effects of acidic sludge addition to the soils on bioavailability and uptake of heavy metals in different parts of pea plant as well as its influence on the growth of that plant. It is observed from our result the abundances of total and bio-available heavy metals in sludge vary as follows: Fe>Mn>Cr>Ni>Cu>Pb>Zn>Cd and Fe>Ni>Mn>Cr>Cu>Zn>Pb>Cd. Sludge applications increased both the total metals, DTPA-extractable metals and total N in the soils. On the other hand lime application has decreased the bioavailability of heavy metals with no change in total N in sludge amended soils. Organic carbon showed positive correlation with all metals except Zn, Cr and Pb. CEC also showed a strong positive correlation (R(2)>0.7) with the low translocation efficiency of pea plants. The value of translocation factor from shoot to seed was found to be smaller than root to shoot of pea plants. Our study thus shows that pea plants were found to be well adapted to the soil amended with 10% sludge with 0.5% lime treatment, minimizing most of the all metal uptake in the shoot of that plant. So, on the basis of the present study, possible treatment may be recommended for the secure disposal of acidic electroplating sludge.     

Replacement of the active surface of a thermophile protein by that of a homologous mesophile protein through structure-guided 'protein surface grafting'

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.     

Introduction of a thermophile-sourced ion pair network in the fourth beta/alpha unit of a psychophile-derived triosephosphate isomerase from Methanococcoides burtonii significantly increases its kinetic thermal stability

Hyperthermophile proteins commonly have higher numbers of surface ionic interactions than homologous proteins from other domains of life. PfuTIM, a triosephosphate isomerase (TIM) from the hyperthermophile archaeon, Pyrococcus furiosus, contains an intricate network of 4 ion pairs in its 4th beta/alpha unit, (β/α)4, whereas MbuTIM, a triosephosphate isomerase from a psychrophile archaeon, Methanococcoides burtonii, lacks this network. Notably, (β/α)4 is the first element of the structure formed during folding of certain TIM-type (beta/alpha)8 barrel proteins. Previously, we have shown that elimination of PfuTIM's ion pair network in PfuTIM significantly decreases its kinetic structural stability. Here, we describe the reciprocal experiment in which this ion pair network is introduced into MbuTIM, to produce MutMbuTIM. Recombinant MbuTIM displays multi-state unfolding with apparent Tm values of autonomous structural elements approaching, or above, 70 °C, when a temperature scanning rate of 90 °C/h is used. The protein displays significant intrinsic kinetic stability, i.e., there is a marked temperature scan rate-dependence of the Tm values associated with unfolding transitions. The Tm values drop by as much as ~ 10 °C when the temperature scanning rate is lowered to 5 °C/h. MutMbuTIM, incorporating PfuTIM's ion pair network, shows significantly higher apparent Tm values (raised by 4–6 °C over those displayed by MbuTIM). MutMbuTIM also displays significantly higher kinetic thermal stability. Thus, it appears that the thermal stability of triosephosphate isomerase can be increased, or decreased, by either enhancing, or reducing, the strength of ion pair interactions stabilizing (β/α)4, presumably through reduced cooperativity (and increased autonomy) in unfolding transitions.     

SGNH hydrolase-type esterase domain containing Cbes-AcXE2: a novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii

Extremophiles - Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of...     

Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.