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At least 415 records · Page 23Linked to original sources
PubMed1999

Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases.

Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.

Bacteria↗
PubMed2001

A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging.

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30 microM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

Automation↗
PubMedDate not supplied

Engineering cytochrome P-450s: chimeric enzymes.

Cytochrome P-450 isozymes represent a critical component of nature's spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5alpha was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.

Amino Acid Sequence↗
PubMed2003

Automated filter paper assay for determination of cellulase activity.

Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in beta-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and beta-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.

Automation↗
PubMed2000

Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis.

In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated. To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required. We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours. Using a 96-well format, the whole process can be performed automatically by a pipetting robot. Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours. The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates. Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface. From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.

Automation↗
PubMed1997

Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

Bacillus subtilis↗
PubMed2002

Designing proteins that work using recombinant technologies.

Therapeutic proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Fusion proteins bring together functions from two different molecules creating therapeutics with completely novel activities. Protein engineering technologies have relied on rational design, directed evolution, DNA shuffling, RNA-peptide fusion, phage and ribosomal display methods to select out candidate protein forms with the desired therapeutic properties. Engineered site-specific pegylation and glycosylation strategies have improved circulation half-life, reduced immunogenicity and increased protein therapeutic stability. In this review we describe how protein engineering techniques have been used to select out, improve stability and clinical efficacy of protein therapeutics.

Animals↗
PubMed2010

AAV's anatomy: roadmap for optimizing vectors for translational success.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Cystic Fibrosis↗
PubMed1999

Hirudin display on the surface of bacteriophage M13.

Hirudin was fused to the N terminus of M13 minor protein gp3 (197-406) through a linker GGGS by inserting both the hirudin gene and the gp8 signal sequence into the modified phagemid vector pCANTAB 5V to construct pCANTAB 5G8-Hir. The expressed fusion protein was directed by gp8 signal peptide into the periplasm and assembled to the phage particle to form the hirudin-phage. The fusion protein and fusion phage were detected with biotin-thrombin by Western blotting analysis. Antithrombin activity analysis confirmed that the hirudin portion in the fusion protein and fusion phage bear similar native conformation. The successful display of hirudin on the surface of M13 phage laid a sound foundation for the further study on directed evolution of antithrombotic proteins with altered properties.

Antithrombins↗
PubMedDate not supplied

[Adaptive types of prolamins--specialized proteins from cereal seeds (Poaceae Banrh.)].

Amino acid composition of prolamins and the whole seed in representatives of 52 cereal genera and 22 tribes, as well as amino acid composition of seed protein fractions in representatives of 9 cereal genera are presented. In terms of proposed directed evolution of the seed proteins and adaptive role of prolamins in evolution and distribution of cereals, generalized data on their content in seed protein complex, electrophoretic, immunochemical, and amino acid composition of the seeds, we develop the notion of prolamins biochemical specialization and their polyphyletic origin during evolution of cereal seed protein complex. Seven adaptive types of prolamins are recognized: Sasa, Molinia, Chloris, Zingeria, Poa, Triticum, and Panicum types. Adaptive types of prolamins were formed in response to an ecological request of the environment where taxon ancestors existed and a taxon appeared and in relation to phylogenetic history of a taxon and its assignment to particular tribe and subfamily.

Adaptation, Biological↗
PubMed2000

Enzyme Mechanisms for Polycyclic Triterpene Formation.

The mechanisms by which triterpene cyclases transform olefins into complex and biologically important polycyclic products have fueled nearly half a century of intense research. Recent chemical and biological studies, together with previous findings, provide intriguing new insights into the enzymatic mechanism of triterpene formation and form a surprisingly detailed picture of these elegant catalysts. It can be concluded that the role of the oxidosqualene cyclases involves protection of the intermediate carbocation against addition of water or deprotonation by base, thereby allowing the shift of the hydride and methyl groups along a thermodynamically and kinetically favorable cascade. Key questions in the areas of structural biology, site-directed mutagenesis, and directed evolution are apparent, now that the first structure of a triterpene cyclase is known.

Journal Article↗
PubMed2001

[Research on the school of Danxi of the Ming dynasty].

Zhu Danxi, the founder of Danxi School, one of the four major schools of the Jin - Yuan dynasties, was famous for its Yin - nourishing theory. Clinically, it was adept in treating miscellaneous disorders, with the "four - injury theory" as its theory for differentiation of Zheng. The medical thinking of Danxi is quite specific that it exerted its clinical and academic influence mostly in the Ming dynasty, with so many schools followed him. Though research on its origin and evolution, direct followers like Dai Sigong, Wang Lü, Lin Chung et al. not only totally inherited Danxi's medical idea, but also became the receiver of his academic experiences. Indirect followers(study perronally) reached its peak in the Ming dynasty. They inherited Danxi's experience and further developed to supplement his theory. For other physicians partly following Danxi's experience also emerged and applied his academy for further development of medical theory.

China↗
PubMed1992

Luminescent screens for general X-ray diagnostics.

This article gives the main parameters of amplifying screens manufactured in the USSR: the sensitivity, the resolution, the modulation depth, the noise. The technical level of Soviet screens is compared with the level of those manufactured by certain foreign companies. The measures taken to increase the technical level of today's products and the further evolution directions are discussed. Data are given on screen production in the USSR during the last 5 years and their supposed production up to 2000.

Evaluation Studies as Topic↗
PubMedDate not supplied

[Metabolic monitoring of heart surgery patients during the immediate postoperative period].

The situation of aggression is accompanied by metabolic alterations with an important neuro-endocrinal context. Extracorporeal surgery is an especially aggressive situation which involves both surgery and the added metabolic state of hypothermia. In 48 patients subjected to extracorporeal circulation with hypothermia, the metabolic parameters of Oxygen Consumption (VO2), Production of carbon dioxide (VCO2), Respiratory Quotient (RQ) and Energy Expenditure (GE) were studied. It was observed that the patients could be divided into two subgroups, depending on their metabolic response. Studying the RQ parameter, it was seen that this divided patients into two subgroups depending on their posterior evolution. Patients in whom RQ did not exceed 0.8 showed more favourable evolution than those in whom RQ was higher than 0.8. The correct metabolic response was regarded as a parameter indicative of the evolution, directly correlated to the hemodynamic parameters studied. In one patient, creatine phosphate was infused at a dosage of 30 mg/kg for 60 min, and an improvement was noted in GE and Cardiac Index. It was concluded that the intravenous intake of creatine phosphate in patients undergoing extracorporeal surgery improved both metabolic parameters and the myocardiac function.

Adult↗
PubMedDate not supplied

[The thermodynamic direction of biological evolution. The model and the reality].

A macrothermodynamic model of evolution of the supramolecular structures and chemical composition of living objects during ontogenesis and at long-term stages of general biological evolution is presented. A study of quasiclosed (thermodynamically and kinetically) systems, phases of the biomass supramolecular structures, enables a conclusion on the thermodynamic direction of biological evolution to be made. In correspondence with the second principle, this direction leads to variations in the chemical composition and structure of the living systems during their development. Indirect and direct evidence of the trend to a minimal Gibbs specific function of formation of the supramolecular structures in animal tissues during ontogenesis are presented. The conclusion that thermodynamics is the "driving force" of evolution of the biological world is confirmed.

Animals↗
PubMed2001

Frogs without polliwogs: evolution of anuran direct development.

Direct development is the assumption of the adult morphology without progression through an intervening, morphologically distinct, free-living larval phase. We discuss the ecological factors contributing to the evolution of this derived life-history strategy in frogs, and the developmental modifications that facilitate such an unusual mode of embryogenesis. Studies on the Puerto Rican tree frog, Eleutherodactylus coqui, have identified several such modifications, including developmental adaptations for dealing with increased egg size, and loss of tadpole structures. Surprisingly, this direct developer still undergoes a thyroid hormone-dependent metamorphosis, which occurs before hatching. We suggest how the ancestral biphasic developmental pattern may have been rearranged during the evolution of direct development.

Animals↗
PubMed2002

Cell-selfish modes of evolution and mutations directed after transcriptional bypass.

During transcription, prokaryotic and eukaryotic RNA polymerases bypass and misread (transcriptional mutagenesis) several classes of DNA lesions. For example, misreading of 8-OH-dG generates mRNAs containing G to T transversions. After translation, if the mutant protein briefly allowed the cell a growth-DNA replication advantage, then precocious DNA replication would bypass that unrepaired 8-OH-dG and misinsert dA opposite the directing DNA lesion with a higher probability than would be experienced for 8-OH-G lesions at other positions in otherwise identical neighboring cells. Such retromutations would have been tested for their imparted growth advantage as mRNA before they became heritable DNA mutations. The logical properties of a mode of evolution that utilizes directed-retromutagenesis were compared one by one with those of the standard neo-Darwinian mode. The retromutagenesis mode, while minimizing mutational load, is cell-selfish; fitness is for an immediate growth advantage rather than future reproductive potential. In prokaryotes, an evolutionary mode that involves standard Darwinian fitness testing of novel alleles in the genetic background of origin followed by clonal expansion also favors cell-selfish allele combinations when linkage disequilibrium is practiced. For metazoa and plants to have evolved organized tissues, cell-selfish modes of evolution represent systems-poisons that must be totally suppressed. The feedback loops that allow evolution to be cell-serving in prokaryotes are actively blocked in eukaryotes by traits that restrict fitness to future reproductive potential. These traits include (i) delay of fitness testing until after the mutation is made permanently heritable, (ii) diploidy to further delay fitness testing, (iii) segregation of somatic lines from germ lines, (iv) testing of novel alleles against randomized allele combinations constructed by obligate sex, and (v) obligate genetic death to insure that that the most basic systems unit of selfish allele combinatorial uniqueness is the species instead of the cell. The analyses indicate that modes of evolution in addition to our neo-Darwinian one could have existed utilizing known molecular mechanisms. The evolution of multicellularity was as much the discarding of old cell-selfish habits as the acquisition of new altruistic ones.

Alleles↗