A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase

Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.     

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G bindingprotein based upon the B domain of protein A (SpAB) from Staphylococcusaureus were studied by substituting tryptophan residues at strategiclocations within each of the three a-helical regions (al-a3)of the domain. The role of the C-terminal helix, a3, was investigatedby generating two protein constructs, one corresponding to thecomplete SpAB, the other lacking a part of ct3; the Trp substitutionswere made in both one-and two-domain versions of each of theseconstructs. The fluorescence properties of each of the single-tryptophanmutants were studied in the native state and as a function ofguanidine-HCl-mediated unfolding, and their IgG binding activitieswere determined by a competitive enzyme-linked immunosorbentassay. The free energies of folding and of binding to IgG foreach mutant were compared with those for the native domains.The effect of each substitution upon the overall structure andupon the IgG binding interface was modelled by molecular graphicsand energy minimization. These studies indicate that (i) 3 contributesto the overall stability of the domain and to the formationof the IgG binding site in l and 2, and (ii) al unfolds first,followed by 2 and 3 together.     

Synthesis, characterization, and catalytic properties of polysiloxanes with pendant 4-(3-pyridinyl)butyl and 4-(1-oxypyridin-3-yl)butyl groups

New silane monomers with the pendant 4-(3-pyridine)butyl group have been synthesized by hydrosilation of 3-(3-butenyl)pyridine with Me _n Si(OEt)_3-n H (n=0, 1) using a platinum catalyst. Only -addition products were observed. The products were characterized by elemental analysis, infrared,¹H- and¹³C-NMR spectroscopy, and gas chromatography-mass spectrometry. Hydrolysis-polycondensation of the difunctional monomer with a basic catalyst, Me₄NOH, gave a mixture of cyclic oligomers, principally cyclic tetramer, and linear homopolymer. Under similar reaction conditions, the trifunctional monomer gave crosslinked material which was soluble in common organic solvents. The linear homopolymer and crosslinked polymer were trimethylsilyl end-blocked with hexamethyldisilazane. The cyclic and end-blocked polymers were characterized by elemental analysis and spectroscopic methods. Molecular weights of the polymers were obtained by end-group analysis using¹H-NMR spectral data, size exclusion chromatography, and direct insertion-probe mass spectrometry. The cyclic, linear, and crosslinked materials were N-oxidized withm-chloroperoxybenzoic acid and characterized by spectroscopic methods. The polymeric N-oxide derivatives were shown to be effective transacylation catalysts in the synthesis of mixed carboxylic acid anhydrides in immiscible solvents (H₂O/CH₂Cl₂) under phase-transfer conditions. The implications of the results on the mechanism of catalysis are discussed.     

Differential Display of Investigative Behavior Permits Discrimination of Scent Signatures from Familiar and Unfamiliar Socially Dominant Female Marmoset Monkeys (Callithrix jacchus)

The hypothesis was tested that marmosets have differential displays of investigatory behavior towards circumgenital scent marks from socially dominant females based on degree of familiarity. In a series of two-choice behavioral discrimination bioassays, dominant males together with subordinate and dominant females were presented concurrently with scent secretions from familiar versus unfamiliar dominant females, and their investigative behaviors towards the scent were recorded. Test animals directed significantly different amounts of investigative behaviors towards familiar versus unfamiliar scents when the scent stimuli involved complete circumgenital scent marks, glandular secretion only, or urine only. When animals discriminated between the two scent samples, they did so by directing significantly increased amounts of behavior to scent from unfamiliar females. Chemical cues promoting differential displays of behavior to familiar versus unfamiliar scents appeared to be volatile in nature and were effective in the scent mark for at least one day following deposition. The reliable ability of marmosets to consistently discriminate between odors from familiar versus unfamiliar dominant females provides strong circumstantial evidence that individual female marmosets have distinct chemical signatures. The ability of marmosets to discriminate the odor from familiar versus unfamiliar dominant females may be important in the context of intergroup relations.     

Activated gas plasma surface treatment of polymers for adhesive bonding

Polyethylene, polypropylene, poly(vinyl fluoride) (Tedlar), polystyrene, nylon 6, poly(ethylene terephthalate) (Mylar), polycarbonate, cellulose acetate butyrate, and a poly(oxymethylene) copolymer were treated with activated helium and with activated oxygen. Mechanical strengths of adhesive-bonded specimens prepared from treated and from untreated coupons were compared. Polyethylene (PE) and polypropylene (PP) showed the greatest increases in bond strength. Oxygen and helium were both effective with polyethylene, but polypropylene showed no improvement when treated with activated helium. The results with excited helium parallel the effects of ionizing radiation on these two polymers, as does the appearance of unsaturation bands in the infrared (965 cm^?1 in PE, and 887 and 910 cm^?1 in PP). Active nitrogen produced excellent bond strength with polyethylene but not with polypropylene. Of the remaining polymers examined, Tedlar, polystyrene, and nylon 6 showed the greatest improvement in bondability after treatment, and Mylar showed moderate improvement. Polycarbonate, cellulose acetate butyrate, and the poly(oxymethylene) copolymer gave approximately two-fold increases in lap-shear bond strength. In several cases, significant differences in response to time of treatment and type of excited gas were found.     

Experiences from using heat integration software to determine retrofit opportunities within a refinery process

Heat integration techniques can be used to optimize the energy requirement for both new and retrofit plant designs. Software tools for identifying retrofit options are becoming available. This paper reports our experiences from using heat exchanger network (HEN) optimization software for a retrofit case study of an oil refinery process. The HEN optimization software was used to automate the search for the most beneficial retrofit designs following the twostage process proposed by Asante and Zhu. The software provided three potential retrofit designs. Results from this analysis were used as the basis of a rigorous mass and energy balance simulation of the plant. The simulation corroborated the energy savings, but there were some important differences. The simulation required 20% more heat exchange area. Furthermore, the retrofit design involving one topology change was shown to be less economic than an alternative design. These differences are discussed and a revised methodology is proposed.     

Prevention of water pollution problems in mining: The bactericide technology

In pyritic environments, the bacteriaThiobacillus ferrooxidans catalyze acid formation by increasing the oxidation rate of pyrite by a factor of one million. This acid solubilizes metals and pollutes adjacent streams and lands. Bactericide sprays during mining and waste disposal operations attack the source of the problem by preventing acid formation and metals solubilization. Used in conjunction with current water treatment systems, bactericides can dramatically reduce operating costs. Controlled release bactericides contribute to successful reclamation by providing assurance against revegetation failure and post-reclamation water quality problems that can necessitate perpetual water treatment. While inhibitingT. ferrooxidans these organic compounds aid in the establishment of beneficial heterotrophic bacteria which support vegetation. These conditions continue to persist after the bactericide is depleted from the controlled release system. Case Studies I and II show that bactericides inhibit acid generation during hard rock and coal mining operations and they are cost effective. Case Studies III and IV illustrate the improvement in water quality and vegetation after reclamation when controlled release bactericides were used. Economic analyses show cost benefits are achieved when controlled-release bactericides are part of the reclamation plan.     

Sand corrosion,thermal expansion,and ablation of medium- and high-entropy compositionally complex fluorite oxides

Sand corrosion, thermal expansion, and ablation properties of a new class of medium- and high-entropy compositionally complex fluorite oxides (CCFOs) are examined as potential protective coating materials. Five binary oxides were mixed and sintered into dense, single-phase CCFOs of the general formula: [Hf_(1-2x)/3Zr_(1-2x)/3Ce_(1-2x)/3Y_xYb_x]O_2-δ (x = 0.2, 0.074, and 0.029). These CCFOs exhibit decreased molten sand infiltration and interaction at intermediate temperatures (1200-1300°C) in comparison with a cubic yttria-stabilized zirconia (YSZ) reference; however, at higher temperatures, the trend is reversed due to the increased chemical reactivity. The equimolar high-entropy (Hf_0.2Zr_0.2Ce_0.2Y_0.2Yb_0.2)O_2-δ exhibits no grain boundary penetration by molten sand at all examined temperatures (1200°C-1500°C), although reaction and precipitation are significant. Moreover, these CCFOs exhibit higher intrinsic thermal expansion coefficients (CTE) than the YSZ reference, thereby being more compatible with Ni-based superalloys. The 8YSZ-like (Hf_0.284Zr_0.284Ce_0.284Y_0.074Yb_0.074)O_2-δ exhibits the highest CTE in this series of CCFOs due to oxygen clustering effects. Finally, these CCFOs also exhibit lower emissivities and form unique faceted microstructures in ablative environments.     

Temperature and processing effects on lithium ion conductivity of solution-deposited lithium zirconium phosphate (LiZr2P3O12) thin films

Lithium zirconium phosphate (LiZr₂P₃O₁₂) thin films have been prepared on platinized silicon substrates via a chemical solution deposition approach with processing temperatures between 700°C and 775°C. Films that were subject to a single high-temperature anneal were found to crystallize at temperatures above 725°C. Crystallization was observed in films annealed after each deposited layer at 700°C and above. In both cases, grain size was found to increase with annealing temperature. Ion conductivity was found to increase with annealing temperature in singly annealed films. In per-layer annealed films ion conductivity was found to initially increase then decrease with increasing annealing temperature. A maximum ion conductivity of 1.6 × 10⁻⁶ S/cm was observed for the singly annealed 775°C condition, while a maximum ion conductivity of 5.8 × 10⁻⁷ S/cm was observed for the 725°C per-layer annealed condition. These results are consistent with an increasing influence of cross-plane, internal interface resistance and vapor phase carrier loss in the per-layer annealed samples. This work demonstrates that post-deposition processing methods can strongly affect the ion conducting properties of LiZr₂P₃O₁₂ thin films.     

The influence of oxygen vacancies on piezoelectricity in samarium-doped Pb(Mg1/3Nb2/3)O3-PbTiO3 ceramics

A study of 0.71Pb(Mg_1/3Nb_2/3)O₃-0.29PbTiO₃ shows that, when it is doped with 2.5% Sm on the A-site, in addition to an almost threefold increase in piezoelectric charge coefficient and dielectric permittivity, there is a 2 order of magnitude reduction in conductivity, attributed to a decrease in oxygen vacancy concentration. An analysis of the nonlinearity of permittivity with respect to field amplitude shows that both the reversible and irreversible contributions increase significantly with Sm-doping, with simple models showing that these changes are consistent with a reduction in the concentration of dipolar defects that can inhibit both polarization rotation and domain wall translation. Contrary to the argument that doping increases heterogeneity, there is little change in the diffuseness of the peak in permittivity as a function temperature, whilst there is a 15% increase in spontaneous polarization with Sm addition. Through comparison of the Rayleigh law parameters with those published for other piezoelectric materials, it is concluded that a significant contribution to the observed increase in piezoelectric performance due to Sm-doping of PMN-PT is similar in origin to that seen in soft, donor-doped PZT and other conventional piezoelectric ceramics.