RESUMEN
Reduction of double bonds of α,ß-unsaturated carboxylic acids and esters by ene-reductases remains challenging and it typically requires activation by a second electron-withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-crystal structural analysis of profen-bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-profens was established and this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-active (S)-profens.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Oxidorreductasas/metabolismo , Propionatos/química , Antiinflamatorios no Esteroideos/química , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Nicotiana/enzimologíaRESUMEN
The era of synthetic biology heralds in a new, more "green" approach to fine chemical and pharmaceutical drug production. It takes the knowledge of natural metabolic pathways and builds new routes to chemicals, enables nonnatural chemical production, and/or allows the rapid production of chemicals in alternative, highly performing organisms. This route is particularly useful in the production of monoterpenoids in microorganisms, which are naturally sourced from plant essential oils. Successful pathways are constructed by taking into consideration factors such as gene selection, regulatory elements, host selection and optimization, and metabolic considerations of the host organism. Seamless pathway construction techniques enable a "plug-and-play" switching of genes and regulatory parts to optimize the metabolic functioning in vivo. Ultimately, synthetic biology approaches to microbial monoterpenoid production may revolutionize "natural" compound formation.
Asunto(s)
Vías Biosintéticas , Escherichia coli/genética , Mentha/genética , Ingeniería Metabólica/métodos , Monoterpenos/metabolismo , Escherichia coli/metabolismo , Genes de Plantas , Microbiología Industrial/métodos , Mentha/enzimología , Mentha/metabolismo , Familia de Multigenes , Operón , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biología Sintética/métodosRESUMEN
Biocatalysis is a useful tool in the provision of chiral technology and extremophilic enzymes are just one component in that toolbox. Their role is not always attributable to their extremophilic properties; as with any biocatalyst certain other criteria should be satisfied. Those requirements for a useful biocatalyst will be discussed including issues of selectivity, volume efficiency, security of supply, technology integration, intellectual property and regulatory compliance. Here we discuss the discovery and commercialization of an L-aminoacylase from Thermococcus litoralis, the product of a LINK project between Chirotech Technology and the University of Exeter. The enzyme was cloned into Escherichia coli to aid production via established mesophilic fermentation protocols. A simple downstream process was then developed to assist in the production of the enzyme as a genetically modified-organism-free reagent. The fermentation and downstream processes are operated at the 500 litre scale. Characterization of the enzyme demonstrated a substrate preference for N-benzoyl groups over N-acetyl groups. The operational parameters have been defined in part by substrate-concentration tolerances and also thermostability. Several examples of commercial biotransformations will be discussed including a process that is successful by virtue of the enzyme's thermotolerance.
Asunto(s)
Bioquímica/métodos , Amidohidrolasas/química , Reactores Biológicos , Biotecnología/métodos , Biotransformación , Catálisis , Estabilidad de Enzimas , Escherichia coli/metabolismo , Fermentación , Calor , Modelos Químicos , Especificidad por Sustrato , Thermococcus/enzimologíaRESUMEN
Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P(1) site, such as valine, alanine or phenylalanine. The K(m) and k(cat) of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca(2+) ions. At 70 degrees C, a 10-fold increase in Ca(2+) concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca(2+) to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca(2+)-binding site in Ak.1 protease. Other metal ions, such as Sr(2+), increase the thermostability of the enzyme, but to a significantly lower degree than does Ca(2+). The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.
Asunto(s)
Disulfuros/metabolismo , Subtilisinas/aislamiento & purificación , Estabilidad de Enzimas , Calor , Conformación Proteica , Especificidad por Sustrato , Subtilisinas/química , Subtilisinas/metabolismoRESUMEN
Proteins of the subtilisin superfamily (subtilases) are widely distributed through many living species, where they perform a variety of processing functions. They are also used extensively in industry. In many of these enzymes, bound calcium ions play a key role in protecting against autolysis and thermal denaturation. We have determined the crystal structure of a highly thermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247), reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third calcium ion, however, is at a novel site that is created by two key amino acid substitutions near Ca-1, and has not been observed in any other subtilase. This site, acting cooperatively with Ca-1, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the mesophilic subtilisins also point to the importance of aromatic clusters, reduced hydrophobic surface and constrained N and C termini in enhancing the thermostability of thermitase and Ak.1 protease. The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that modifies the active site cleft geometry.
Asunto(s)
Bacillus/enzimología , Calcio/metabolismo , Subtilisinas/química , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Bacillus/genética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Subtilisinas/genética , TemperaturaRESUMEN
The gene for a highly thermostable neutral proteinase (Npr) was isolated from Bacillus sp. strain EA1 by the polymerase chain reaction using consensus primers based on the sequences of npr genes from related species. The gene was sequenced and shown to be closely related to a neutral proteinase gene from Bacillus caldolyticus strain YP-T; the mature form of the enzyme differing by only a single amino acid. Enzyme samples were prepared from both the native organisms and also from recombinant Escherichia coli expressing the two npr genes. The proteinase from strain EA1 was shown to be significantly more thermostable than that from B. caldolyticus and that this difference is the result of a single amino acid substitution which is situated proximal to a region of the enzyme known to be crucial to conferring thermal stability. The phylogenetic relationship of EA1 to other Bacilli is also described.
Asunto(s)
Bacillus/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Bacillus/clasificación , Bacillus/enzimología , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Calor , Concentración de Iones de Hidrógeno , Metaloendopeptidasas/biosíntesis , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoAsunto(s)
Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Archaea/enzimología , Archaea/genética , Ácido Aspártico Endopeptidasas/metabolismo , Bacillus/enzimología , Bacillus/genética , Biotecnología , Calcio/farmacología , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/genética , Estabilidad de Enzimas/efectos de los fármacos , Genes Bacterianos , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Serina Endopeptidasas/metabolismo , Temperatura , Thermus/enzimología , Thermus/genéticaRESUMEN
Bacillus sp. strain Wp22.A1 produced a cell-associated aspartic proteinase which was purified to homogeneity using phenyl-Sepharose (hydrophobic and affinity chromatography) and Mono Q. The proteinase has a molecular mass of 45 kDa by SDS/PAGE and a pI of 3.8. It is insensitive to pepstatin, but is sensitive to the other aspartic proteinase-specific inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane. Inactivation by DAN was only partial, suggesting that it had non-specifically modified an aspartate residue at a site other than the active site. The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested. Maximum proteolytic activity was observed at pH 3.5. The proteinase had a higher activity with haemoglobin, but was more specific (Vmax./Km) for cytochrome c. Substrate inhibition was observed with both these substrates. The cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val2-Asn3. The proteinase was stable in the pH range 2.5-5.5. Thermostability was increased in the presence of Ca2+, although to a lesser extent at higher temperatures. The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+.