The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15–35 % hydrophilic ILs, even at 75–90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15–25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower Vmax. However, only minor changes in kinetic parameter Km showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-016-0841-y) contains supplementary material, which is available to authorized users.

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Unconventional reservoirs associated with the presence of fractures have been identified in the Jurassic horizons in the Raudhatain field in North Kuwait. These reservoirs contain high-quality light oil and gas under HPHT conditions with associated H2S and CO2. The operator in this field is presently developing these gas/light oil reserves under the ‘North Kuwait Gas Development Project, The well discussed in this paper is one of a number of new production wells. Historically, Jurassic wells in this field have been drilled using conventional barite-weighted, oil-based mud (OBM) with densities up to 18.5 ppg. However, the challenge to maximize the amount and quality of reservoir data collected and to reduce any impact on the formation caused by OBMs led to a search for an optimized water-based mud (WBM) system as an alternative. A WBM formulated with saturated potassium-formate brine, weighted with manganese tetra-oxide, was selected for the following characteristics: higher base-fluid density, non-damaging nature, robust behavior in HPHT environments, and the possibility of better quality image logs. The well reached 16,530 ft., in a 6–in. hole, with bottomhole temperatures around 280°F. The fluid was thermally resistant, exhibiting very low HPHT filtrate loss and highly stable rheological properties. The maximum fluid density was 16.2 ppg, with no evidence of settling of weight material, even after three days of static conditions. A complete suite of logs was run successfully. The elemental capture spectroscopy (ECS), along with sonic and neutron porosity logs provided good data quality, and the image logs showed excellent resolution. This paper will present all aspects of the planning, design, and utilization of this new drilling fluid. It will focus on lessons learned and conclude with recommendations for further optimization of the design of these drilling fluids.

Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO could also be utilized in the conversion of CO to useful products via formate (HCOO). In this study, we investigated the reduction of CO in the form of hydrogen carbonate (HCO) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO reduced the reaction rate. CtFDH was modeled in the presence of HCO showing that it fits to the active site. The active site setting for hydride transfer in CO reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.