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PubMed2000

Unnatural ligands for engineered proteins: new tools for chemical genetics.

Small molecules that modulate the activity of biological signaling molecules can be powerful probes of signal transduction pathways. Highly specific molecules with high affinity are difficult to identify because of the conserved nature of many protein active sites. A newly developed approach to discovery of such small molecules that relies on protein engineering and chemical synthesis has yielded powerful tools for the study of a wide variety of proteins involved in signal transduction (G-proteins, protein kinases, 7-transmembrane receptors, nuclear hormone receptors, and others). Such chemical genetic tools combine the advantages of traditional genetics and the unparalleled temporal control over protein function afforded by small molecule inhibitors/activators that act at diffusion controlled rates with targets.

Alleles↗
PubMed2001

Microfabricated reaction and separation systems.

Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated 'lab-on-a-chip' systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.

Automation↗
PubMed2002

Wrapping and inclusion of organic molecules with ultrathin, amorphous metal oxide films.

In this review, we overview metal oxide nanostructures in which organic molecules play important roles as templates, as structural units, and, in some cases, as hosts. Their structural precision and diversity are discussed from the viewpoint of the topology of a metal-oxygen network. Supramolecular capsules of metal oxides are prepared by the self-assembly of polyoxometalates. Zeolites and mesoporous materials are synthesized by using organic molecules with their assemblies acting as templates. The topological networking of silsesquioxanes makes it possible to produce novel nanocomposites and microporous materials. In the final section, we demonstrate our recent studies into molecular imprinting, the encapsulation of a fluorescent dye, and the wrapping of individual polymer chains. Ultrathin, amorphous metal oxide films can retain the shape of organic molecules and can be used to create molecular composites by precisely wrapping individual molecules. These films are also effective in insulating molecular functions from external environments. The advantages of amorphous metal oxides are discussed in relation to the properties of the corresponding crystalline metal oxides and their potential prospects in nanotechnology.

Binding Sites↗
PubMed2001

Physiology, regulation, and limits of the synthesis of poly(3HB).

The properties of poly(3-hydroxybutyrate) combined with the fact that it can be produced easily by numerous prokaryotes from renewable resources and even from potentially toxic waste products using well-known fermentation processes have generated keen interest in this biopolyester as a substitute for chemo-synthetic petroleum-derived polymers in many applications. However, the high price of poly(3HB) compared with the conventional synthetic materials currently in use has restricted its availability in a wide range of applications. If the economic viability of poly(3HB) production and its competitiveness are to be improved, more must be found out about the phenotypic optimization and the upper limits of bacterial systems as the factory of poly(3HB). In this chapter, two aspects of poly(3HB) are reviewed--poly(3HB) formation as a physiological response to external limitations and overcoming internal bottlenecks, and poly(3HB) as a commercially attractive polyester. From a physiological viewpoint, the ability to synthesize and degrade poly(3HB) is considered an investment in the future and provides organisms with a selective advantage. Poly(3HB) is presented as a strategic survival polymer, and it is shown that growth-associated synthesis is not as rare as reported. The influence of the efficiency and velocity of cell multiplication and product formation, of poly(3HB) content and of productivity on the overall yield, and finally on the economics of the whole process are discussed and evaluated from the technological or consumer's point of view. The specific production rate and poly(3HB) content appear to be more important than the yield coefficients.

Chemical Engineering↗
PubMed2001

Production of microbial polyesters: fermentation and downstream processes.

Poly(3-hydroxyalkanoates) (PHAs) constitute a large and versatile family of polyesters produced by various bacteria. PHAs are receiving considerable attention because of their potential as renewable and biodegradable plastics, and as a source of chiral synthons since the monomers are chiral. Industrial PHA production processes have been developed for poly(3-hydroxybutyrate) (poly(3HB)) and poly(3-hydroxybutyrate-co-3-valerate) (poly(3HB-co-3HV). More than 100 other poly(3HAMCL)s, characterized by monomers of medium chain length, have been identified in the past two decades. These monomers typically contain 6-14 carbon atoms, are usually linked via-3-hydroxy ester linkages, but can occasionally also exhibit 2-, 4-, 5-, or 6-hydroxy ester linkages. Such polyesters are collectively referred to as medium chain length PHAs poly(3HAMCL)s. The vast majority of these interesting biopolyesters have been studied and produced only on the laboratory scale. However, there have been several attempts to develop pilot scale processes, and these provide some insight into the production economics of poly(3HAMCL)s other than poly(3HB) and poly(3HB-co-3HV). These processes utilize diverse fermentation strategies to control the monomer composition of the polymer, enabling the tailoring of polymer material properties to some extent. The best studied of these is poly(3-hydroxyoctanoate) (poly(3HO)), which contains about 90% 3-hydroxyoctanoate. This biopolyester has been produced on the pilot scale and is now being used in several experimental applications.

Chemical Engineering↗
PubMed1999

The FMN to heme electron transfer in cytochrome P450BM-3. Effect of chemical modification of cysteines engineered at the FMN-heme domain interaction site.

The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3 (Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., and Poulos, T. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1863-1868) indicates that the proximal side of the heme domain molecule is the docking site for the FMN domain and that the Pro(382)-Gln(387) peptide may provide an electron transfer (ET) path from the FMN to the heme iron. In order to evaluate whether ET complexes formed in solution by the heme and FMN domains are structurally relevant to that seen in the crystal structure, we utilized site-directed mutagenesis to introduce Cys residues at positions 104 and 387, which are sites of close contact between the domains in the crystal structure and at position 372 as a control. Cys residues were modified with a bulky sulfhydryl reagent, 1-dimethylaminonaphthalene-5-sulfonate-L-cystine (dansylcystine (DC)), to prevent the FMN domain from binding at the site seen in the crystal structure. The DC modification of Cys(372) and Cys(387) resulted in a 2-fold decrease in the rates of interdomain ET in the reconstituted system consisting of the separate heme and FMN domains and had no effect on heme reduction in the intact heme/FMN-binding fragment of P450BM-3. DC modification of Cys(104) caused a 10-20-fold decrease in the interdomain ET reaction rate in both the reconstituted system and the intact heme/FMN domain. This indicates that the proximal side of the heme domain molecule represents the FMN domain binding site in both the crystallized and solution complexes, with the area around residue 104 being the most critical for the redox partner docking.

Bacillus megaterium↗
PubMed2005

State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET+. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.

Adenosine Triphosphate↗
PubMed1989

Patent literature as a source of information for research and development: an investigation on calcium phosphate-containing biomaterials, Part I.

The usefulness of patent literature for research and development is mostly unknown. Therefore a specific patent retrieval has been carried out concerning calcium phosphate-containing biomaterials. This research field includes chemical, medical, and engineering problems and is of importance to the development of bioactive materials for bone replacement. The preliminary work includes information on the characteristics and the availability of patent literature as well as about patent classification systems according to which the documents are filed in patent collections. By reading the non-patent literature searching questions can be formulated. The proper patent retrieval starts with the study of secondary literature especially that in Chemical Abstracts, which report on patents since 1907. The structure of Chemical Abstracts, their indexes and sections help to find relevant patents of chemical or chemical engineering contents fast and inspire to read patents of bordering areas. This retrieval from Chemical Abstracts led to 171 patents disclosed 1975-1985 and to 95 patents disclosed in 1986; the latter are listed in a table. The contents of the abstracts inform on the research activity and help to reduce time and effort for a continuation of the retrieval in a patent collections or database.

Bibliographies as Topic↗
PubMed1999

Chemical exposures of rocket-engine test-stand personnel and cancer mortality in a cohort of aerospace workers.

We conducted a retrospective cohort study of 6107 aerospace workers to examine whether exposure to chemicals--primarily hydrazine fuels--during rocket-engine fueling and testing affects cancer mortality. When conditional logistic regression analysis was applied and adjusted for confounding variables, the estimated rate ratio for lung cancer mortality, comparing exposed to unexposed workers from the same facility, ranged from 1.68 (95% confidence interval, 1.12 to 2.52) to 2.10 (95% confidence interval, 1.36 to 3.25), depending on job-duration threshold (6 or 24 months) and lag (0 to 15 years). Similar results were obtained for hemato- and lymphopoietic cancer and for bladder and kidney cancer mortality, but estimates for these cancers were imprecise. We concluded that occupational exposure to hydrazine or other chemicals associated with rocket-engine testing jobs increased the risk of dying from lung cancer, and possibly other cancers, in this population of aerospace workers; however, our results need to be replicated in other populations.

Carcinogens↗
PubMed2001

A continuous membrane bioreactor for ester synthesis in organic media: II. Modeling Of MBR continuous operation.

A model was developed to describe the conversion degree in a membrane bioreactor (MBR) for the synthesis of short-chain esters as a function of the flow rate. The transesterification reaction was catalyzed by a recombinant cutinase of Fusarium solani pisi microencapsulated in reversed micelles of AOT/isooctane. The differences of product concentration in permeate and retentate together with the deactivation profiles led to an enzyme distribution evaluation that describes the experimental values attained. The model considers the bioreactor design as well as its hydrodynamics and the enzyme kinetics. The approach included the analysis of the MBR operation as a CSTR, a PFR, and a series of continuous reactors. The comparative efficiency of these reactor types is discussed. The enzyme distribution was estimated for all the cases. The best description was obtained considering a series of two CSTRs. The modeling results led to a re-evaluation of cutinase operational stability. Deactivation rates correlated very well with the hydrodynamic aspects of biocatalyst location.

Bioreactors↗
PubMed1990

Interfacial mass transfer in extraction of amino acid by ALIQUAT 336 in organic phase.

Reactive extraction of L-phenylalanine from alkaline aqueous solution into xylene in the presence of tri-octyl-methyl-ammonium chloride (ALIQUAT 336) as complexing agent was studied using a stirred transfer cell. The study investigated the effects of carrier concentration and temperature on mass transfer rates. Transfer rate across the interface in the presence of surfactant molecules was also studied. A two-film model was proposed by considering film mass transfer resistances at the aqueous and organic phases. The model predicted adequately the experimental time-concentration data at different carrier concentrations and temperatures. The model was modified to take into account the presence of surfactant in the organic phase.

Chemical Engineering↗
PubMed2001

In vitro biosynthesis of polyesters.

In vitro synthesis of polyesters using isolated enzymes as catalyst via non-biosynthetic pathways is reviewed. In most cases, lipase was used as catalyst and various monomer combinations, typically oxyacids or their esters, dicarboxylic acids or their derivatives/glycols, and lactones, afforded the polyesters. The enzymatic polymerization often proceeded under mild reaction conditions in comparison with chemical processes. By utilizing characteristic properties of lipases, regio- and enantioselective polymerizations proceeded to give functional polymers, most of which are difficult to synthesize by conventional methodologies.

Chemical Engineering↗
PubMed2003

Know-how and know-why in biochemical engineering.

This contribution analyzes the position of biochemical engineering in general and bioprocess engineering particularly in the force fields between fundamental science and applications, and between academia and industry. By using culture technology as an example, it can be shown that bioprocess engineering has moved slowly but steadily from an empirical art concerned with mainly know-how to a science elucidating the know-why of culture behavior. Highly powerful monitoring tools enable biochemical engineers to understand and explain quantitatively the activity of cellular culture on a metabolic basis. Among these monitoring tools are not just semi-online analyses of culture broth by HPLC, GC and FIA, but, increasingly, also noninvasive methods such as midrange IR, Raman and capacitance spectroscopy, as well as online calorimetry. The detailed and quantitative insight into the metabolome and the fluxome that bioprocess engineers are establishing offers an unprecedented opportunity for building bridges between molecular biology and engineering biosciences. Thus, one of the major tasks of biochemical engineering sciences is not developing new know-how for industrial applications, but elucidating the know-why in biochemical engineering by conducting research on the underlying scientific fundamentals.

Biochemistry↗