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Results for “Directed Molecular Evolution”

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At least 847 records · Page 47Linked to original sources
PubMed2001

Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40.

The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene. We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two. Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon. If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations. If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions. We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present. We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.

Carotenoids↗
PubMed1998

Spectroscopic and functional studies of a novel quadruple myoglobin variant with increased peroxidase activity.

A quadruple variant of horse heart myoglobin (Thr39Ile/Lys45Asp/Phe46Leu/Ile107Phe) that exhibits significantly (approximately 25-fold) greater peroxidase activity than the wild-type protein has been studied to determine its midpoint reduction potential (24(2) mV vs. SHE; pH 6.0, mu = 0.1 M, 25 degrees C) and to characterize the kinetics of its reaction with hydrogen peroxide. In addition, Fourier transform infrared (FTIR) spectra of the carbonyl and azide adducts of the protein have been obtained to gain initial insight into the effects of these substitutions on the ligand binding properties of the reduced and oxidized variant. All of the results obtained in this work are consistent with a variant heme binding pocket with increased hydrophilic character.

Animals↗
PubMed2000

Protein engineering of subtilisin.

The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.

Calcium↗
PubMed2000

Biotechnology in the 21st century.

Although the future is unpredictable, it is highly likely that biotechnology will play a much more visible and significant role in the 21st century than it did in the 20th century. The number and kinds of drugs provided by biotechnology will expand markedly and biotechnology will stand at the center of the oncoming revolution in bioinformatics.

Biotechnology↗
PubMed2000

Reinventing the wheel.

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Directed Molecular Evolution↗
PubMed2002

Microbial technologies for the discovery of novel bioactive metabolites.

Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development and application of molecular methods for the detection of uncommon genera of actinomycetes in soil DNA and for the rapid dereplication of actinomycete isolates. The results indicate a substantial presence in many soils of the uncommon genera and a large diversity of isolated actinomycetes. However, while uncommon actinomycete strains may provide an increased chance of yielding novel structures, their genetics and physiology are poorly understood. To speed up their manipulation, we have developed vectors capable of stably maintaining large segments of actinomycete DNA in Escherichia coli and of integrating site specifically in the Streptomyces genome. These vectors are suitable for the reconstruction of gene clusters from smaller segment of cloned DNA, the preparation of large-insert libraries from unusual actinomycete strains and the construction of environmental libraries.

Actinobacteria↗
PubMed2001

Novel concepts for selection of catalytic activity.

The selection of mutant enzymes with novel properties from libraries is emerging as a very powerful strategy for enzyme engineering. The past year has witnessed significant progress on several fronts: new and improved methods have been developed for the creation of libraries and advances have been made in screening and selection techniques. The results achieved demonstrate the enormous potential of the methods and leave questions open for further studies.

Catalysis↗
PubMed2001

In vitro display technologies: novel developments and applications.

In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.

Binding Sites↗
PubMed2002

Towards novel processes for the fine-chemical and pharmaceutical industries.

In response to the need in the pharmaceutical industry for more complex, chiral molecules, fine-chemical companies are embracing new manufacturing technologies to produce compounds of these specifications. In particular, recent developments in biocatalysis combined with novel process engineering are providing improved methods for the production of valuable chemical intermediates.

Catalysis↗
PubMed1998

Expression of ribozymes in gene transfer systems to modulate target RNA levels.

The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.

Animals↗
PubMed2000

Biotechnology match making: screening orphan ligands and receptors.

To date there has been a considerable amount of interest and success in the pharmaceutical industry in the discovery of drug targets and diagnostics via genomic technologies, namely DNA sequencing, mutation/polymorphism detection and expression monitoring of mRNA. As the ultimate targets for the majority of these methods are actually proteins, more and more emphasis has been placed upon protein-based methods in an effort to define the function of proteins discovered by genomic technologies. One of the most challenging areas of drug target discovery facing researchers today is the search for novel receptor-ligand pairs. Database mining techniques in conjunction with other computational methods are able to identify many novel sequences of putative receptors, but the ability to similarly identify the receptor's natural ligand is not possible by these methods. The past few years have seen an increase in methodology and instrumentation focused on the ability to discover and characterize protein-protein interactions, as well as receptor-ligand pairs. Significant advances have been made in the areas of instrumentation (biosensors and fluorescent plate readers) as well as methodologies relating to phage/ribosome display and library construction.

Animals↗
PubMed2002

Fitness effects of fixed beneficial mutations in microbial populations.

Beneficial mutations are intuitively relevant to understanding adaptation, yet not all beneficial mutations are of consequence to the long-term evolutionary outcome of adaptation. Many beneficial mutations-mostly those of small effect-are lost due either to (1) genetic drift or to (2) competition among clones carrying different beneficial mutations, a phenomenon called the "Hill-Robertson effect" for sexual populations and "clonal interference" for asexual populations. Competition among clones becomes more prevalent with increasing genetic linkage and increasing population size, and it is thus generally characteristic of microbial populations. Together, these two phenomena suggest that only those beneficial mutations of large fitness effect should achieve fixation, despite the fact that most beneficial mutations produced are predicted to have very small fitness effects. Here, we confirm this prediction-both empirically and theoretically-by showing that fitness effects of fixed beneficial mutations follow a distribution whose mode is positive.

Adaptation, Physiological↗
PubMed1999

The counterforce.

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Directed Molecular Evolution↗