SEARCH · PubMed Health

Results for “enzyme evolution”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed2002

Molybdenum and tungsten in biology.

Molybdenum is the only second-row transition metal that is required by most living organisms, and the few species that do not require molybdenum use tungsten, which lies immediately below molybdenum in the periodic table. Because of their unique chemical versatility and unusually high bioavailability these two transition metals have been incorporated into the active sites of enzymes over the course of evolution. Enzymes that contain molybdenum or tungsten continue to be discovered and several crystal structures have become available recently. This new structural information has been complemented by spectroscopic and kinetic methods, as well as computational approaches. Together, these studies provide an increasingly detailed view of the reaction mechanisms and the correlation between the electronic structure of the active site and catalytic function, one of the fundamental goals in metallobiochemistry.

Aldehyde Oxidoreductases↗

PubMed2001

Directed Evolution and Biocatalysis.

This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction. This technology offers the opportunity to adapt the enzyme to the needs of the process. We describe herein the development of enzyme evolution technology and particularly DNA shuffling. We also consider several classes of enzymes, their current applications, and the limitations that should be addressed. In a review of this length it is impossible to describe all the enzymes with potential for industrial exploitation; there are other classes, which given appropriate activity, selectivity, and robustness, could become useful tools for the industrial chemist. This is an exciting era for biocatalysis and we expect great progress in the future.

Journal Article↗

PubMed1995

Enzymes: chemistry and biochemistry.

Basic principles underlying enzyme action are considered. Catalytic antibodies (abzymes), catalytic RNA (ribozymes), and non-biological counterparts of enzyme-catalyzed reactions are mentioned. Enzyme evolution is considered in terms of divergence, convergence, and lateral gene transfer.

Amino Acid Sequence↗

PubMed2000

A novel biotinylated suicide inhibitor for directed molecular evolution of lipolytic enzymes.

A bifunctional activity label (8) for directed molecular evolution of lipolytic enzymes has been designed and synthesized. The structure is composed of a 4-nitrophenyl activated phosphonate, that is, a suicide substrate of lipases/esterases, connected to a biotin moiety through a spacer containing a disulfide bridge. The phosphonate (3) was prepared by Michaelis-Arbuzov reaction of trimethylsilyl-protected 11-bromoundecanol (2) with triethyl phosphite. The deprotected omega-hydroxyalkylphosphonate (4) was transformed into an active N-hydroxysuccinimide carbonate (5) followed by 4-nitrophenyl activation of the phosphonate using standard procedures. The biotinylated phosphonate inhibitor (8) was then synthesised by coupling the phosphonate inhibitor (6) to the epsilon-amino-caproic acid and cystamine containing biotinyl spacer (7). The function of all relevant groups of the final activity label (8) (biotin-label, cleavable disulfide bridge, phosphonate-inhibitor) have been successfully tested with the commercial lipase Lipolase (Novo Nordisk). Hence, a tool for directed molecular evolution of lipolytic enzymes has been developed.

Affinity Labels↗

PubMed2000

[Molecular modification and artificial evolution of microbial enzymes].

There exists a remarkably progress in molecular modification and artificial evolution of microbial enzymes in recent years. Error-prone polymerase chain reaction and DNA shuffling have been developed and successfully applied and a lot of startling achievements have been obtained in artificial evolution of enzymes.

Bacteria↗

PubMed1997

Selective neutrality and enzyme kinetics.

This article appeals to a recent theory of enzyme evolution to show that the properties, neutral or adaptive, which characterize the observed allelic variation in natural populations can be inferred from the functional parameters, substrate specificity, and reaction rate. This study delineates the following relations between activity variables, and the forces--adaptive or neutral--determining allelic variation: (1) Enzymes with broad substrate specificity: The observed polymorphism is adaptive; mutations in this class of enzymes can result in increased fitness of the organism and hence be relevant for positive selection. (2) Enzymes with absolute substrate specificity and diffusion-controlled rates: Observed allelic variation will be absolutely neutral; mutations in this class of enzymes will be either deleterious or have no effect on fitness. (3) Enzymes with absolute or group specificity and nondiffusion-controlled rates: Observed variation will be partially neutral; mutants which are selectively neutral may become advantageous under an appropriate environmental condition or different genetic background. We illustrate each of the relations between kinetic properties and evolutionary states with examples drawn from enzymes whose evolutionary dynamics have been intensively studied.

Adaptation, Physiological↗

PubMed2004

Directed evolution of nucleic acid enzymes.

Just as Darwinian evolution in nature has led to the development of many sophisticated enzymes, Darwinian evolution in vitro has proven to be a powerful approach for obtaining similar results in the laboratory. This review focuses on the development of nucleic acid enzymes starting from a population of random-sequence RNA or DNA molecules. In order to illustrate the principles and practice of in vitro evolution, two especially well-studied categories of catalytic nucleic acid are considered: RNA enzymes that catalyze the template-directed ligation of RNA and DNA enzymes that catalyze the cleavage of RNA. The former reaction, which involves attack of a 2'- or 3'-hydroxyl on the alpha-phosphate of a 5'-triphosphate, is more difficult. It requires a comparatively larger catalytic motif, containing more nucleotides than can be sampled exhaustively within a starting population of random-sequence RNAs. The latter reaction involves deprotonation of the 2'-hydroxyl adjacent to the cleavage site, resulting in cleaved products that bear a 2',3'-cyclic phosphate and 5'-hydroxyl. The difficulty of this reaction, and therefore the complexity of the corresponding DNA enzyme, depends on whether a catalytic cofactor, such as a divalent metal cation or small molecule, is present in the reaction mixture.

DNA, Catalytic↗

PubMed2004

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.

Bacteria↗

PubMed2005

How to broaden enzyme substrate specificity: strategies, implications and applications.

For identification of mutations associated with the broadening of enzyme substrate specificity, three strategies, including directed enzyme evolution, are described for selected examples. Implications concerning enzyme models are highlighted. Applications to the field of biocatalysis are discussed. A bidimensional map for the classification of enzyme activities is suggested so as to improve genome annotations.

Catalysis↗

PubMed2005

High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS). Earlier in vivo studies have demonstrated that Ebg, a protein of unknown function, can evolve to allow Escherichia coli lacking the lacZ beta-galactosidase gene to grow on lactose. Here we demonstrate that we can evolve Ebg into an enzyme with significant beta-galactosidase activity in vitro. Only two specific mutations were ever seen to provide this improvement in Ebg beta-galactosidase activity in vivo. In contrast, nearly all the improved beta-galactosidases selected in vitro resulted from different mutations.

Directed Molecular Evolution↗

PubMed2006

Analysis of the exon-intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei.

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution.

Amino Acid Sequence↗

PubMed2001