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Background Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2–C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil–water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase—Thermomyces lanuginosus (TLIP). Results TON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni–NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (Topt). The far UV–CD spectrums were consistent with a well-folded α/β-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates. Conclusions Here, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals. Electronic supplementary material The online version of this article (10.1186/s13068-019-1452-5) contains supplementary material, which is available to authorized users.

Commercial applications of β-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 ± 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 ± 0.21 pNP U/ml with specific activity of 35.52 ± 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme.

Glucose inhibition of β-glucosidase (BG) is a bottleneck in biomass hydrolysis. In this study, a glucose resistant GH1 β-glucosidase gene- Mbgl from Methylococcus capsulatus (bath strain) was cloned and overexpressed in E.coli. The Ni-NTA affinity purified Mbgl displayed an optimum temperature of 70°C and optimum pH was 6.0. The calculated KM of the enzyme was 48.6mM and 0.12mM for cellobiose and 4-Nitrophenyl β-d-glucopyranoside (PNPG) respectively. PNPG hydrolysis in presence of various glucose concentrations showed that the enzyme was stimulated by ∼2.2 fold at 50mM glucose and was not inhibited up to 450-500mM glucose. Homology modeling and structural comparisons of Mbgl with a glucose tolerant β-glucosidase of Humicola insolens (HiBG) revealed that the Mbgl has a much broader active site unlike to a deep and narrow active site pocket of HiBG. The difference in active site shape reflects on an alternative mechanism of glucose tolerance in Mbgl. Supplementing a commercial cellulase enzyme mixture CTec with Mbgl in the hydrolysis of the pretreated rice straw enhanced the glucose yield by 10-15%. In addition, Mbgl was also stable in organic solvents, detergents and oxidative conditions which would be advantageous for biotechnological applications.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (H(2)), obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H(2) production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O(2) sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H(2) with the physiological electron donor, reduced ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.

) is the most abundant pentose in ourplanet.Itisamajorbuildingblockofhemicellulose,oneofthemain constituents of lignocellulosic biomass. Xylose com-prises ca. 20–30% weight of plant matter. Because it is noteconomically feasible to separate xylose from other biomasssugars, the co-utilization of pentose and hexose is essential tothe success of the production of biocommodities frombiomass. In principle, one mole of xylose when combinedwith H

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

We investigate here the high structural stability of Pyrococcus furiosus triosephosphate isomerase (PfuTIM) by exploring the effects - upon the protein's structure and kinetic thermal stability - of modulation of its ionic interactions through pH variations, and mutations. PfuTIM shows comparable structural contents at pH 3.0, 7.0 and 10.0. However, at pH 3.0, subtle changes are seen in the protein's surface hydrophobicity and association status, and its kinetic thermal stability is profoundly reduced (as evidenced by its facile heat- and cold-mediated denaturation, characterized by a high degree of hysteresis and irreversibility). Increase in ionic strength through addition of salt counters the reduction of stability, and reversal of pH facilitates partial refolding. Further, a mutated form of PfuTIM (mPfuTIM) lacking 4 key charged residues involved in ionic interactions displays a structural content identical to PfuTIM but profound reduction in kinetic stability to thermal and chemical denaturation, as well as evidence of partial unfolding at temperatures between 90 degrees C and 100 degrees C, unlike PfuTIM. We conclude, therefore, that ionic interactions (which are known to determine protein thermodynamic stability) can also contribute significantly to protein kinetic thermal stability.

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 degrees 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 degrees C. Remarkably, over 50% activity is retained at 70 degrees 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.

The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE.

The genome of Methylococcus capsulatus (bath) encodes a protein R-est6 that is annotated as a lipase family 3 protein. The phylogenetic and the sequence analyses linked this protein to the family 6 carboxylesterase. The gene encoding R-est6 was cloned and overexpressed in Escherichia coli and the recombinant 6x-His tagged protein was purified by Ni-NTA affinity chromatography. The buffers used in the purification were modified by adding 1% glycerol instead of the salt to prevent the protein aggregation. Far UV-CD spectrum and gel filtration chromatography of the purified R-est6 confirmed that the protein was well folded like a typical α/β hydrolase and had the quaternary structure of a tetramer, in addition to a compact monomer. The optimum pH was in the range of 7.0-9.0 and the optimum temperature was at 55 °C for the hydrolysis of pNP-butyrate. As expected, being a member of the family 6 carboxylesterase, R-est6 hydrolyzed triglycerides, pNP esters of the small and the medium fatty acid chain esters and an aryl ester-phenyl acetate. However, R-est6 was also found to hydrolyze the long-chain fatty acid ester which had never been reported for the family 6 carboxylesterase. Additionally, R-est6 was stable and active in the different water-miscible organic solvents. Therefore, the broad substrate range and the structural stability of R-est6 would be advantageous for its application in industrial processes.

The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.

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.

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as ‘Trishanku’ unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native ‘Trishanku’ refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.

Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to15 enzymes under mild reaction conditions (i.e. 37 °C and atm). In a batch reaction, the hydrogen yield was 23.2 mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74 mmol of H 2 /L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.

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.

Cold denaturation is a phenomenon seen in many different proteins. However, there have been no reports so far of its occurrence in hyperthermophile proteins. Here, using a recombinant triosephosphate isomerase (PfuTIM) from the hyperthermophile archaeon, Pyrococcus furiosus, we show that the heating of this protein through the low temperature side of its thermal unfolding transition in the presence of guanidinium hydrochloride (GdmCl) results in the formation of partially-disordered conformational ensembles that retain considerable native-like secondary and tertiary structure. Unlike PfuTIM itself, these thermochemically obtained partially-disordered PfuTIM ensembles display cold denaturation as they are cooled to room temperature. The protein thus shows hysteresis, adopting different structural states in a manner dependent upon the nature of the heating and cooling treatment, rather than upon the initial and final conditions of temperature and GdmCl concentration, indicating that some sort of a kinetic effect influences structure adoption and retention. The structure lost through cooling of partially-disordered PfuTIM is found to be regained through heating. The ability of GdmCl to thus apparently destabilize the highly thermodynamically and kinetically stable structure of PfuTIM (sufficiently, to cause it to display observable cold-denaturation and heat-renaturation transitions, in real-time, with cooling and heating) offers support to current ideas concerning the how hyperthermophile proteins achieve their high kinetic stabilities, and suggests that desolvation-solvation barriers may be responsible for high kinetic stability.

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.

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.

BACKGROUND: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. METHODOLOGY: We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Abeta(1-42)) peptide is modified. Changes in the hydrodynamic volume of Abeta(1-42) in the presence of arginine measured by size exclusion chromatography show that arginine binds to Abeta(1-42). Arginine increases the solubility of Abeta(1-42) peptide in aqueous medium. It decreases the aggregation of Abeta(1-42) as observed by atomic force microscopy. CONCLUSIONS: Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

The data presented in this article are connected to the research article entitled “Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath)” (Soni et al., 2016 [1]). The family 6 carboxylesterases are the smallest and display broad substrate specificity. The 1kb gene encoding, a family 6 carboxylesterase – R-est6, was amplified from the genome of M. capsulatus (bath strain), and showed in the agarose gel. The corresponding purified protein, after overexpression in Escherichia coli, was biochemically studied in the research article (Soni et al., 2016 [1]). R-est6 has hydrophobic patches on the surface so, it is expected to show oligimeric forms. Here, we have confirmed the presence of oligomers by gel filtration chromatography data and the proteins belonging to the different peaks are shown on a SDS-PAGE.