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PubMed2005

Testing the trend towards specialization in herbivore-host plant associations using a molecular phylogeny of Tomoplagia (Diptera: Tephritidae).

Herbivorous insects are abundant and diverse and insect-host plant associations tend to be specialized and evolutionarily conserved. Some authors suggested that generalist insect lineages tend to become specialists, with host specialization leading to an evolutionary dead-end for the parasite species. In this paper, we have examined this tendency using a phylogenetic tree of Tomoplagia (Diptera: Tephritidae), a parasite of asteracean plants. We have tested the trend towards specialization in different hierarchical degrees of host specialization. The topology of the tree, the inference of ancestral hosts, and the lack of directional evolution indicated that specialization does not correspond to a phylogenetic dead-end. Although most Tomoplagia species are restricted to a single host genus, specialization does not seem to limit further host range evolution. This work emphasizes the advantages of the use of different levels of specialization and the inclusion of occasional hosts to establish a more detailed scenario for the evolution of this kind of ecological association.

Adaptation, Biological↗
PubMed2002

Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein.

A novel assay, referred to as fluorophor-linked immunosorbent assay (FLISA), for the characterization of single-chain antibody fragments (scFvs) is described. The principle of the method is the fusion of an scFv to enhanced green fluorescent protein (EGFP). The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain. The time-consuming binding of secondary antibodies and enzyme reaction, necessary for enzyme-linked immunosorbent assays (ELISAs) are not required. Consequently, the assay time of 1.5 h needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 h. This renders the FLISA suitable for applications where a short assay time is essential, such as screening of mutant libraries of scFvs in directed evolution experiments or monitoring of the amount of functionally expressed recombinant protein during production processes. In contrast to a comparable ELISA, the FLISA showed no saturation when determining the relative amount of functional scFv. The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R(2)>0.99. The usefulness of a competitive FLISA for the detection of analytes is shown exemplarily by the detection of s-triazines with the s-triazine-specific scFv K411B.

Antibody Specificity↗
PubMed1998

Overexpression, immobilization and biotechnological application of Pseudomonas lipases.

Pseudomonas lipases play an important role in biotechnology both as hydrolases for detergent additives and as synthases catalyzing the kinetic resolution of racemic compounds. Large-scale production of Pseudomonas lipases requires correct folding and secretion through the bacterial membranes. Controllable expression of the gene lipH encoding a lipase-specific foldase proves to be important for overexpression in the homologous host Escherichia coli. Construction of appropriate His-tagged fusion proteins permitted overexpression, secretion and one-step purification of lipase from culture supernatants of the homologous host Pseudomonas aeruginosa. The immobilization of lipases in hydrophobic sol-gel materials derived from alkylsilane precursors of the type RSi(OCH3)3 or mixtures of RSi(OCH3)3 and Si(OCH3)4 provides highly active chemically and thermally stable heterogeneous biocatalysts. The entrapped lipases are excellent catalysts in a variety of synthetic organic transformations. Using directed evolution based on error prone PCR, the enantioselectivity of the hydrolysis of a chiral ester, catalyzed by the lipase from P. aeruginosa, can be increased from ee 2 to ee 81% in just four mutagenesis cycles.

Bacterial Proteins↗
PubMed2004

Yeast mating for combinatorial Fab library generation and surface display.

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

Antibody Affinity↗
PubMed1999

Derivation of RNA aptamer inhibitors of human complement C5.

Specific aptamer inhibitors of the human complement C5 component were produced by the SELEX methodology of directed evolution of nucleic acid ligands. The SELEX procedure started with a pool of random-sequence, 2'F-pyrimidine-modified nuclease-stabilized RNA, and after twelve rounds of iterative C5 binding and nucleic acid amplification an evolved RNA pool was obtained which contained the highest affinity binders to the C5 protein. The evolved RNA pool was then cloned and sequenced, and individual clones were analyzed for binding and function. Twenty-eight clones (out of sixty) were identified which bound C5 (termed aptamers). Seven of these aptamers formed a closely related sequence homology family; these aptamers bound C5 with a Kd 20-40 nM and also inhibited human serum hemolytic activity. In addition, these aptamers inhibited zymosan-induced generation of C5a. Aptamer inhibition of both C5b and C5a suggests that aptamer binding inhibits cleavage of C5 by the C5 convertase of both pathways. One of the inhibitory aptamer sequences was truncated to yield a 38-mer 2'F RNA aptamer which retained C5 binding and inhibitory activity. The structure of this aptamer is predicted to be a stem-loop containing thirteen base pairs, and also containing two bulges. The affinity of this aptamer was improved by performing a second biased SELEX experiment, where the randomized starting RNA pool uses a template where the individual base compositions are biased toward a specific sequence. This second SELEX experiment produced an aptamer with a Kd of 2-5 nM which retained functional activity. Another SELEX to rat C5 produced an aptamer with binding and inhibitory properties virtually identical with the human aptamer. The human and rat aptamers are being evaluated for complement inhibition in vitro and in vivo as potential therapeutics for treatment of human disease.

Animals↗
PubMed2000

The consensus concept for thermostability engineering of proteins.

Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The 'consensus approach' described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal phytases was used to calculate a consensus phytase amino acid sequence. Upon construction of the synthetic gene, recombinant expression and purification, the first phytase obtained, termed consensus phytase-1, displayed an unfolding temperature (T(m)) of 78.0 degrees C which is 15-22 degrees C higher than the T(m) values of all parent phytases used in its design. Refinement of the approach, combined with site-directed mutagenesis experiments, yielded optimized consensus phytases with T(m) values of up to 90.4 degrees C. These increases in T(m) are due to the combination of multiple amino acid exchanges which are distributed over the entire sequence of the protein and mainly affect surface-exposed residues; each individual substitution has a rather small thermostabilizing effect only. Remarkably, in spite of the pronounced increase in thermostability, catalytic activity at 37 degrees C is not compromised. Thus, the design of consensus proteins is a potentially powerful and novel alternative to directed evolution and to a series of rational approaches for thermostability engineering of enzymes and other proteins.

6-Phytase↗
PubMed2003

'Super bugs' for bioremediation.

Chlorinated organic compounds are among the most significant pollutants in the world. Sequential use of anaerobic halorespiring bacteria, which are the key players in biological dehalogenation processes, and aerobic bacteria whose oxygenases are modified by directed evolution could lead to efficient and total degradation of highly chlorinated organic pollutants. Recently three interesting papers on halorespiration and polychlorinated biphenyl biodegradation were published.

Bacteria, Anaerobic↗
PubMed2002

Phage display selects for amylases with improved low pH starch-binding.

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.

Amino Acid Sequence↗
PubMed2003

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor.

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Bacillus Phages↗
PubMed2004

Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops.

Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 degrees C, and lost activity with a half-life of 30 min at 80 degrees C in the presence of 10 mM CaCl2.

Amino Acid Sequence↗
PubMed2002

Developments and trends in enzyme catalysis in nonconventional media.

The conventional notion that enzymes are only active in aqueous media has long been discarded, thanks to the numerous studies documenting enzyme activities in nonaqueous media, including pure organic solvents and supercritical fluids. Enzymatic reactions in nonaqueous solvents offer new possibilities for producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug molecules, biopolymers, peptides and proteins, modified fats and oils, structured lipids and flavor esters). The use of enzymes in both macro- and microaqueous systems has been investigated especially intensively in the last two decades. Although enzymes exhibit considerable activity in nonaqueous media, the activity is low compared to that in water. This observation has led to numerous studies to modify enzymes for specific purposes by various means including protein engineering. This review covers the historical developments, major technological advances and recent trends of enzyme catalysis in nonconventional media. A brief description of different classes of enzymes and their use in industry is provided with representative examples. Recent trends including use of novel solvent systems, role of water activity, stability issues, medium and biocatalyst engineering aspects have been discussed with examples. Special attention is given to protein engineering and directed evolution.

Journal Article↗
PubMed1997

Monoclonal gammopathy of undetermined significance evolving directly in primary plasma cell leukemia.

A case of monoclonal gammopathy of undetermined significance (MGUS) which evolved directly into primary plasma cell leukemia (PCL) is reported. Even if MGUS and PCL represent the opposite aspects of the same immunoproliferative disorder, no previous cases of this direct evolution have been described, as stated by an accurate review of the specific literature. The patient abruptly developed PCL after five months from the discovery of a serum monoclonal spike, without any other clinical, laboratory and radiological abnormalities. The practical conclusions of this case report indicate that a patient with MGUS must be followed periodically and over an indefinite period, and that PCL must be considered into the possible evolution of MGUS.

Humans↗
PubMed2000

Biodesulfurization and the upgrading of petroleum distillates.

Biotechnology offers an alternative way to process fossil fuels. There have been several important advances in the elucidation of the mechanisms of biodesulfurization and the development of a biocatalytic desulfurization process. These include a detailed analysis of the rate and extent of desulfurization of real target molecules in a diesel matrix, the directed evolution of rate- and extent-limiting enzymes for better performance and the expression of the genes in alternative hosts. Process innovations include new reactor designs, separations and recovery strategies and the production of value-added byproducts during desulfurization.

Bacteria↗
PubMed2001

Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids.

A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.

Amidohydrolases↗
PubMed2002

Industrial enzyme applications.

The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used.

Agriculture↗
PubMed2002

Methods to increase enantioselectivity of lipases and esterases.

Lipases and esterases are frequently used in the synthesis of optically pure compounds; however, natural enzymes do not always show sufficiently high enantioselectivity. Variation of the structure of the substrates, modification of the reaction system or protein engineering (e.g. the expression of pure enzymes, rational design or directed evolution) are strategies that can be employed to improve the distinction between two enantiomers or enantiotopic groups.

Enzyme Activation↗
PubMed1997

Protein engineering from a bioindustrial point of view.

Work with proteins, particularly enzymes, is a rapidly growing segment of the biotechnology industry. Directed evolution promises to become an increasingly important strategy in their development as it allows one to sidestep some of the difficult questions relating the structural and functional properties of such proteins to their industrial utility. It is also clear, however, that greater understanding of how to engineer certain basic enzyme properties, such as stability, activity, and surface properties, is beginning to emerge, and this understanding will make rational design more efficient. To engineer a commercially useful protein many properties need to be changed, and frequently these changes are interdependent. Recent protein engineering studies on protease, amylase, lipase and cellulase illustrate some of the progress in this area.

Journal Article↗
PubMed2000

Towards a circuit engineering discipline.

Genetic circuits can now be engineered that perform moderately complicated switching functions or exhibit predictable dynamical behavior. These 'forward engineering' techniques may have to be combined with directed evolution techniques to produce robustness comparable with naturally occurring circuits.

Escherichia coli↗