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PubMed2004

Recent advances in enzyme assays.

Enzyme assays for high-throughput screening and enzyme engineering, which are often based on derivatives of coumarin, nitrophenol, fluorescein, nitrobenzofurazane or rhodamine dyes, can be divided into two categories: those that depend on labelled substrates, and those that depend on sensing the reactions of unmodified substrates. Labelled substrates include, for example, fluorogenic and chromogenic substrates that generate a reporter molecule by beta-elimination, fluorescence resonance energy transfer (FRET) substrates and isotopic labels for enantioselectivity screening. By contrast, endpoint sensing can be done using amine reagents, fluorescent affinity labels for phosphorylated proteins, or synthetic multifunctional pores. Sensing assays can also be done in real time by using, for example, aldehyde trapping to follow vinyl ester acylation in organic solvent or calcein-copper fluorescence for sensing amino acids. The current trend is to assemble many such assays in parallel for enzyme profiling and enzyme fingerprinting.

Biochemistry↗

PubMed2005

Site-directed mutations (Asp405Ile and Glu124Ile) in cytochrome P450scc: effect on adrenodoxin binding.

Cytochrome P450scc, mitochondrial adrenodoxin (Adx), and adrenodoxin reductase (AdR) are an essential components in a steroid hydroxylation system. In particular, mytochondrial cytochrome P450scc enzyme catalyses the first step in steroid hormones biosynthesis, represented by the conversion of cholesterol to pregnenolone. In order to study the effect of single mutations on the Adx binding a model of bovine cytochrome P450scc, previously optimized by molecular modeling, was utilized. It was hypothesized by molecular docking that two residues (Asp405 and Glu124) are involved in Adx binding. By site-directed mutagenesis, two mutants of cytochrome P450scc (Asp405Ile and Glu124Ile) expressed in Escherichia coli, were realized by replacing with isoleucines. The site-directed mutations effect on Adx binding was evaluated by differential spectral titration. The apparent dissociation constant values for Asp405Ile and Glu124Ile cytochrome P450scc show that the mutated residues seem to be at the interaction domain with Adx or at least close to it, as predicted by molecular modeling study. Finally, the engineered enzymes were characterized by biochemical and biophysical techniques such as circular dichroism (CD), UV/Vis spectroscopy, and electrochemical analysis.

Adrenodoxin↗

PubMed2001

Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms.

Methods to microencapsulate enzyme, cells, and genetically engineered cells have been described in this article. More specific examples of enzyme encapsulation include the microencapsulation of xanthine oxidase for Lesch-Nyhan disease; phenylalanine ammonia lyase for pheny, ketonuria and microencapsulation of multienzyme systems with cofactor recycling for multistep enzyme conversions. Methods for cell encapsulation include the details for encapsulating hepatocytes for liver failure and for gene therapy. This also includes the details of a novel two-step method for encapsulation of high concentrations of smaller cells. Another new approach is the detailed method of the encapsulation of genetically engineered Escherichia coli DH5 cells for lowering urea, ammonia, and other metabolites in kidney or, liver failure and other diseases.

Animals↗

PubMed1986

Primary structure of Mucor miehei aspartyl protease: evidence for a zymogen intermediate.

The gene encoding the aspartyl protease of the filamentous fungus Mucor miehei has been cloned in Escherichia coli and the DNA sequenced. The deduced primary translation product contains an N-terminal region of 69 amino acid (aa) residues not present in the mature protein. By analogy to the evolutionarily related mammalian gastric aspartyl proteases it is inferred that the primary secreted product is a zymogen containing a 47-aa propeptide. This propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. To study the effects of modifications of the protease structure on its maturation by enzyme-engineering methods, an efficient expression system was sought. In E. coli, transcription of the preproenzyme coding sequence from a bacterial promoter results primarily in the accumulation of unsecreted, enzymatically inactive polypeptides, immunologically related to the authentic protease. In Aspergillus nidulans expression of the cloned gene, probably from its own promoter, results in the secretion into the culture medium of polypeptides which, compared to the authentic protease, are similar in specific activity, but differ in the character of their asparagine-linked oligosaccharides.

Amino Acid Sequence↗

PubMed2005

The selective cause of an ancient adaptation.

Phylogenetic analysis reveals that the use of nicotinamide adenine dinucleotide phosphate (NADP) by prokaryotic isocitrate dehydrogenase (IDH) arose around the time eukaryotic mitochondria first appeared, about 3.5 billion years ago. We replaced the wild-type gene that encodes the NADP-dependent IDH of Escherichia coli with an engineered gene that possesses the ancestral NAD-dependent phenotype. The engineered enzyme is disfavored during competition for acetate. The selection intensifies in genetic backgrounds where other sources of reduced NADP have been removed. A survey of sequenced prokaryotic genomes reveals that those genomes that encode isocitrate lyase, which is essential for growth on acetate, always have an NADP-dependent IDH. Those with only an NAD-dependent IDH never have isocitrate lyase. Hence, the NADP dependence of prokaryotic IDH is an ancient adaptation to anabolic demand for reduced NADP during growth on acetate.

3-Isopropylmalate Dehydrogenase↗

PubMed1999

Separation and direct detection of raw and gelatinized starch hydrolyzing activities of glucoamylase on isoelectric focusing gels.

A procedure to detect raw and gelatinized starch activities of glucoamylase on isoelectric focusing (IEF) gels by using 2, 3, 5-triphenyltetrazolium chloride is described. The reagent reacts with the reducing group of glucose released by glucoamylase from the substrate starch. Using the reaction, production of glucoamylase by Aspergillus niger was detected on 10% IEF gels within a pH range of 2.5-9.5. Since the method can detect raw and gelatinized starch activities of glucomylase associated with 1 microg protein, it will be useful for enzyme engineering studies that involve screening of various mutations.

Aspergillus niger↗

PubMed2007

Novel enzymes through design and evolution.

The generation of enzymes with new catalytic activities remains a major challenge. So far, several different strategies have been developed to tackle this problem, including site-directed mutagenesis, random mutagenesis (directed evolution), antibody catalysis, computational redesign, and de novo methods. Using these techniques, a broad array of novel enzymes has been created (aldolases, decarboxylases, dehydratases, isomerases, oxidases, reductases, and others), although their low efficiencies (10 to 100 M(-1) s(-l)) compared to those of the best natural enzymes (10(6) to 10(8) M(-1) s(-1)) remains a significant concern. Whereas rational design might be the most promising and versatile approach to generating new activities, directed evolution seems to be the best way to optimize the catalytic properties of novel enzymes. Indeed, impressive successes in enzyme engineering have resulted from a combination of rational and random design.

Catalysis↗

PubMed2026

Microblasting Wound Dressings Mechanically Disrupt Polymicrobial Biofilms to Enhance Healing in Treatment-Resistant Wounds.

Treatment-resistant wounds driven by polymicrobial biofilms are a major clinical challenge, affecting millions globally and leading to chronic inflammation, persistent pain, and poor healing outcomes. These wounds are characterized by mature biofilms reinforced by dense extracellular polymeric substances, which confer strong tolerance to conventional treatments. Despite emerging technologies, such as nanoparticles, bacteriophages, and engineered enzymes, effective clearance of established biofilms remains challenging. Here, we develop a microblasting wound dressing (µBLAST) that delivers spatially confined mechano-chemical disruption at the tissue-biofilm interface to remove viscoelastic biofilm matrices and promote tissue regeneration. The µBLAST is assembled by embedding MnO2-doped diatom biosilica beneath an H2O2-releasing cellulose mesh, enabling localized catalytic microbubble generation within biofilm matrices. Confined expansion and rupture of oxygen bubbles produce localized mechanical stress sufficient to dislodge mature, antibiotic-resistant polymicrobial biofilms, while sustained H2O2 release prolongs particle activity. In a murine wound model infected with mature P. aeruginosa and methicillin-resistant S. aureus biofilms, µBLAST treatment significantly reduces biofilm burden, accelerates re-epithelialization, promotes hair regrowth, and mitigates inflammation. Moreover, µBLAST enhances antibiotic efficacy, suppressing biofilm regrowth even at ten-fold reduced drug doses. These findings highlight confined mechano-chemical biofilm disruption as a therapeutic strategy for treating mature, antibiotic-resistant biofilm infections and promoting tissue regeneration.

Biofilms↗

PubMed2000

Residue 285 in cytochrome P450 2B4 lacking the NH(2)-terminal hydrophobic sequence has a role in the functional association of NADPH-cytochrome P450 reductase.

Cytochrome P450 2B4 (CYP2B4) lacking the NH(2)-terminal signal anchor sequence (2-27) was used to study the impact of replacement of histidine with alanine at position 285 on electron transfer from NADPH-cytochrome P450 reductase (P450R). Absorption and circular dichroism spectra of the recombinant hemoproteins indicated that amino acid substitution neither grossly perturbed the geometry of the immediate heme vicinity nor the global polypeptide backbone folding. Fitting of the initial-velocity patterns of P450R-directed reduction of the ferric CYP2B4 (2-27) forms to the Michaelis-Menten kinetics revealed an approximately 3.5-fold increase in the apparent K(m) value for the electron donor of the H285A mutant, while its reductive capacity (V(max)) remained unchanged; this caused a strong drop in reductive efficiency of the engineered enzyme. Circumstantial analysis suggested that impaired association of the redox partners accounted for this phenomenon. Thus, deletion of the positive charge at position 285 of CYP2B4 (2-27) might have disrupted contacts with oppositely charged entities on the P450R surface. Measurements of the stoichiometry of aerobic NADPH consumption and H(2)O(2) production disclosed the oxyferrous H285A species to autoxidize more readily compared with the shortened wild type. This was assumed to arise from less efficient coupling of the system due to defective donation of the second electron by P450R. These results are consistent with the view that His-285 in the truncated CYP2B4 is of importance in the functional interaction with the flavoprotein reductase.

Amino Acid Substitution↗

PubMed2000

Molecular organization of protein-DNA complexes for cell-targeted DNA delivery.

Multifunctional proteins are interesting candidates for nonviral gene transfer to and expression in their target cells. Since at difference of viral vectors, the performance of these vehicles depends on their functional optimisation, a better comprehension of the molecular organisation within protein-DNA complexes would be of great help in reaching their full delivery potential. In this work, we have characterised an RGD-tagged, cell-targeted multifunctional beta-galactosidase carrying a poly-lysine-based DNA-binding domain. In solution, the engineered enzyme spontaneously forms proteinaceous particles of between 20 and 40 nm in diameter that might contain around 10 molecules of enzymatically active protein. Plasmid DNA is efficiently condensed into these particles without modification of the shape, morphology or enzymatic activity, indicative of a comfortable molecular accommodation to the DNA-binding domains. Although the RGD peptide remains equally solvent-exposed and immunoreactive at different DNA-protein ratios, an optimal expression level of cell-delivered genes and integrin-binding specificity are both achieved at 0.02 microg of DNA per microg of protein, indicative of influences of the packaged nucleic acid on the interaction between filled vehicles and the receptors of target cells.

DNA↗

PubMed1998

Structure-function relationships of mitochondrial monoamine oxidase A and B: chimaeric enzymes and site-directed mutagenesis studies.

To gain insight into the structure of monoamine oxidases (MAO) A and B, we investigated the properties of various chimaeric enzymes, engineered by moving progressively the junction between the NH2- and the COOH-termini of each MAO form. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimaeras with increasing length of the NH2-terminus of MAO-A (up to position 256) showed a marked decrease in affinity towards substrates and inhibitors. Two sequences, spanning position 62 to 103 and 146 to 220, appeared of particular importance in putatively constituting the binding site of MAO-B. Conversely, the catalytic properties and specificity of MAO-A were insensitive to substitution of both the NH2-(up to position 112) and COOH-termini (from residue 395), but further modification of the central sequence of MAO-A was not compatible with activity. None of the engineered chimaeras showed a shift in substrate and inhibitor specificity. Investigation on MAO-B by site-directed mutagenesis revealed that His382 and Thr158 may represent residues relevant for MAO-B catalytic mechanism.

Amino Acid Substitution↗

PubMed2006

Orthric Rieske dioxygenases for degrading mixtures of 2,4-dinitrotoluene/naphthalene and 2-amino-4,6-dinitrotoluene/4-amino-2,6-dinitrotoluene.

Pollutants are frequently found as mixtures yet it is difficult to engineer enzymes with broad substrate ranges on aromatics. Inspired by the archetypal nitroarene dioxygenase, which shares its electron transport with a salicylate monooxygenase, we have created an innovative and general approach to expand the substrate range of dioxygenase enzymes in a single cell. We have developed here a series of novel, hybrid dioxygenase enzymes that function with a single ferredoxin reductase and ferredoxin that are used to transport two electrons from nicotinamide adenine dinucleotide to the two independent terminal oxygenases. Each independent alpha-oxygenase may then be used simultaneously to create orthric enzymes that degrade mixtures of environmental pollutants. Specifically, we created a hybrid dioxygenase system consisting of naphthalene dioxygenase/dinitrotoluene dioxygenase to simultaneously degrade 2,4-dinitrotoluene and naphthalene (neither enzyme alone had significant activity on both compounds) and dinitrotoluene dioxygenase/nitrobenzene dioxygenase to simultaneously degrade the frequently encountered 2,4,6-trinitrotoluene reduction products 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene.

Aniline Compounds↗

PubMed2001

Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones.

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.

Burkholderia cepacia↗

PubMed2001

Biotechnology and genetics of ergot alkaloids.

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.

Biotechnology↗

PubMed1994

Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases.

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase, Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.

Base Sequence↗

PubMed2004

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues.

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.

Amino Acid Substitution↗

PubMed1999

Nucleic acid molecular switches.

Natural and artificial ribozymes can catalyse a diverse range of chemical reactions. Through recent efforts in enzyme engineering, it has become possible to tailor the activity of ribozymes to respond allosterically to specific effector compounds. These allosteric ribozymes function as effector-dependent molecular switches that could find application as novel genetic-control elements, biosensor components or precision switches for use in nanotechnology.

Allosteric Regulation↗

PubMed1996

Dental observations in Gaucher's disease: review of the literature and two case reports with 13- and 60-year follow-ups.

Gaucher's disease is a genetically determined inborn error of metabolism in which acid beta-glucosidase or one of its cofactors is absent or diminished in macrophage cells and cause an accumulation of glucosylceramide in these cells. These Gaucher cells accumulate in the organs of the reticuloendothelial system and cause varying degrees of splenomegaly, hepatomegaly, and encroachment of the marrow cavity of bones. Dental radiographs often reveal consistent bony changes that result from this encroachment. In cases where visceral signs are not apparent, dental radiographs can detect the presence of the disease. Because therapy is available, early recognition of this disorder may reduce overall morbidity. A review of the dental radiographic changes over as many as 60 years shows the effects of oral surgery procedures on bone degeneration and regeneration. The effects of mannose lectin acid beta-glucosidase, alglucerase on bone deposition are discussed. Finally, a literature review shows that the changes in the dental radiograph of patients with Gaucher's disease are very specific. Currently, this disorder and its associated molecular genetics are a prototype for research of new treatments such as allogenic bone marrow transplantation and molecularly engineered enzymes.

Adult↗