Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low krel/Kapp for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.

A prediction model has been developed for submerged arc weld-metal chemical composition in terms of flux ingredients with the help of statistical experiments for mixture (extreme vertices design). Bead-on-plate weld deposits as per statistical mixture design experiments were performed at the following welding parameters: current (400 A), voltage (26 V), speed (4.65 mm/s) and electrode extension (30 mm) using CaO-MgO-CaF 2 -Al 2 O 3 flux system. The results show that some of the individual flux ingredients and their binary mixtures have a predominant effect on weld-metal oxygen, manganese, silicon, sulphur, nickel and carbon content. The predicted results show a reasonably good agreement with the experimental results, which were obtained by performing the actual experiments based on a randomly designed flux. Analysis of the experimental data indicate that several mechanisms such as basicity index, oxygen potential, oxide stability, viscosity, electrode chemical reaction, kinetics of slag-metal reaction, etc. are operating simultaneously to yield the final weld-metal composition.

There is growing interest in developing non-photosynthetic routes for the conversion of CO2 to fuels and chemicals. One underexplored approach is the transfer of energy to the metabolism of genetically modified chemolithoautotrophic bacteria. Acidithiobacillus ferrooxidans is an obligate chemolithoautotroph that derives its metabolic energy from the oxidation of iron or sulfur at low pH. Two heterologous biosynthetic pathways have been expressed in A. ferrooxidans to produce either isobutyric acid or heptadecane from CO2 and the oxidation of Fe(2+). A sevenfold improvement in productivity of isobutyric acid was obtained through improved media formulations in batch cultures. Steady-state efficiencies were lower in continuous cultures, likely due to ferric inhibition. If coupled to solar panels, the photon-to-fuel efficiency of this proof-of-principle process approaches estimates for agriculture-derived biofuels. These efforts lay the foundation for the utilization of this organism in the exploitation of electrical energy for biochemical synthesis.

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The prediction model has been developed for low carbon steel weld metal acicular ferrite microstructure as a function of flux ingredients such as CaO, MgO, CaF 2 and Al 2 O 3 in submerged arc welding carried out at fixed welding parameters. The results of quantitative measurements of acicular ferrite (AF) on eighteen no. of weld metal samples were utilised for developing the prediction model. Among the flux ingredients, CaO appears to be most important as an individual as well as interaction with other ingredients in controlling the amount of acicular ferrite content in the weld metal. Furthermore, formation of acicular ferrite is also related to the weld bead geometry which is influenced by flux ingredients. The prediction equation for acicular ferrite has been checked for adequacy by performing separate experiments on welding using randomly designed flux. The isoresponse curves were developed to show the level of acicular ferrite content at different percentage of flux ingredients.KEY WORDS: submerged arc welding; statiscal experiment for mixture; extreme vertices design; flux ingredients; synergism; antagonism; carbon equivalent; inclusion characteristics; weld bead parameter.plex phenomenon with multiple factors and mechanisms operating simultaneously. The target structure and properties are commonly obtained by adjusting variables such as current, voltage, speed and wire-flux combination by trial and error. Since flux plays an important role in the final weld metal composition and inclusion characteristics in SAW process, understanding the flux composition and microstructure of the weld metal remains a major objective in producing maximum amount of acicular ferrite and thereby optimum mechanical properties.The aim of the present work is to predict acicular ferrite content of the microstructure of weld metal, made with different flux ingredients in CaO-MgO-CaF 2 -Al 2 O 3 flux system using given filler wire and welding parameters in order to provide a rational basis for choosing flux composition. This is important in order to establish flux formulation for optimum mechanical properties. Planning of the ExperimentsThe Idea of this present set of investigations is to take account simultaneous variation of each flux ingredient in a flux mixture in order to find out the effect of each flux ingredient as well as their interaction effects on weld metal microstructural constituent acicular ferrite. This has been made possible by applying "statistical design of experiment" involving mixtures, in particular "extreme vertices design", according to which proportions of the important flux ingredients were varied simultaneously to investigate the simultaneous effect of each flux ingredient and their interactions on weld metal microstructure in CaO-MgOCaF 2 -Al 2 O 3 flux system. · Extreme Vertices Design AlgorithmIn experiments using mixture of q components, the components are expressed as a fraction x of the total mixture and the response is a function of the proportions of the components and not the total amount of mix...

The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.

A series of diaroyl phosphates was employed to assess the general reactivity of this class of molecule against classical class A and class C β-lactamases. The compounds were found, in general, to be inhibitory substrates of both classes of enzyme. In each case, they reacted rapidly with the enzyme (10 4 -10 6 s −1 M −1 ) to yield transiently stable intermediates, most likely acyl-enzymes, which slowly (10 −3 -10 −1 s −1 ) regenerated free enzyme. In certain cases, side branches from direct turnover produced EII complexes ("substrate" inhibition), more inert EI′ complexes, and, in one case, a completely inactive EI′ complex. Deacylation, but not acylation, was enhanced by electronwithdrawing substituents. Acylation rates were enhanced by hydrophobic substitution, both in the diaroyl phosphate and at the enzyme active site. The latter factor led to the general order of β-lactamase acylation rates: class D (previous results) > class C > class A. It is likely that nanomolar inhibitors of all serine β-lactamases could be achieved by rational exploitation of diacyl phosphates.The threat of pathogenic bacteria to human health continues to rise. Such bacteria are now often resistant to many, if not all, of the antibiotics that we routinely use to defend ourselves from them (1). Among those antibiotics whose effectiveness is threatened by the spread of bacterial resistance are the β-lactams, which have been in constant clinical use since 1945, and which are probably the generally most effective and safe class of such compounds. Although there are many sources of bacterial resistance to β-lactams, the most frequently encountered derives from the β-lactamases, bacterial enzymes that catalyze the hydrolysis of β-lactams and thus destruction of their antibiotic activity (2).Resistance to β-lactam antibiotics arising from β-lactamases can, both in principle and in practice, be reduced by the administration of effective β-lactamase inhibitors together with β-lactams (3). Therefore, just as the search for new antibiotics continues, so too does the search for new β-lactamase inhibitors (4,5). It is now very well established that there are a large number of β-lactamases, differing in active site structure and substrate specificity, and spanning four molecular homology classes A-D (6). It is unlikely that a common and yet potent non-covalent inhibitor can be found to span all of these enzymes. Experience with class A, C and D β-lactamases, the serine enzymes, has shown that covalent inhibitors of the mechanism-based variety and transition state analogue inhibitors (also most likely acting covalently since the chemical transition states are covalently bound to the enzymes), aimed at the reaction center, are most likely to be broadly successful.Our studies of phosphorus-based inhibitors of serine β-lactamases have demonstrated two important points. First, that anionic phosphylating agents (1) can be effective inhibitors (7), and second, that negatively charged phosphate leaving groups †