RESUMO
Aromatic L-amino acid decarboxylases (AADCs) catalyze the conversion of aromatic L-amino acids into aromatic monoamines that play diverse physiological and biosynthetic roles in living organisms. For example, dopamine and serotonin serve as major neurotransmitters in animals, whereas tryptamine and tyramine are essential building blocks for synthesizing a myriad of secondary metabolites in plants. In contrast to the vital biological roles of AADCs in higher organisms, microbial AADCs are found in rather a limited range of microorganisms. For example, lactic acid bacteria are known to employ AADCs to achieve intracellular pH homeostasis and engender accumulation of tyramine, causing a toxic effect in fermented foods. Owing to the crucial pharmaceutical implications of aromatic monoamines and their derivatives, synthetic applications of AADCs have attracted growing attention. Besides, recent studies have uncovered that AADCs of human gut microbes influence host physiology and are involved in drug availability of Parkinson's disease medication. These findings bring the bacterial AADCs into a new arena of extensive research for biomedical applications. Here, we review catalytic features of AADCs and present microbial applications and challenges for biotechnological exploitation of AADCs. KEY POINTS: ⢠Aromatic monoamines and their derivatives are increasingly important in the drug industry. ⢠Aromatic L-amino acid decarboxylases are the only enzyme for synthesizing aromatic monoamines. ⢠Microbial applications of aromatic L-amino acid decarboxylases have drawn growing attention.
Assuntos
Descarboxilases de Aminoácido-L-Aromático , Carboxiliases , Aminoácidos Aromáticos , Animais , Descarboxilases de Aminoácido-L-Aromático/química , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Serotonina/metabolismo , Tiramina/metabolismoRESUMO
Aromatic L-amino acid decarboxylases (AADCs) are ubiquitously found in higher organisms owing to their physiological role in the synthesis of neurotransmitters and alkaloids. However, bacterial AADC has not attracted much attention because of its rather limited availability and narrow substrate range. Here, we examined the biochemical properties of AADC from Bacillus atrophaeus (AADC-BA) and assessed the synthetic feasibility of the enzyme for the preparation of monoamine neurotransmitters. AADC-BA was expressed in Escherichia coli BL21(DE3) and the purified enzyme showed a specific activity of 2.6 ± 0.4 U/mg for 10 mM L-phenylalanine (L-Phe) at 37 °C. AADC-BA showed optimal pH and temperature ranges at 7-8 and 37-45 °C, respectively. The KM and kcat values for L-Phe were 7.2 mM and 7.4 s-1, respectively, at pH 7.0 and 37 °C. Comparison of the kinetic constants at different temperatures revealed that the temperature dependency of the enzyme was mainly determined by catalytic turnover rather than substrate binding. AADC-BA showed a broad substrate scope for various aromatic amino acids, including L-Phe, L-tryptophan (610% relative to L-Phe), L-tyrosine (12%), 3,4-dihydroxyphenyl-L-alanine (24%), 5-hydroxy-L-tryptophan (L-HTP, 71%), 4-chloro-L-phenylalanine (520%), and 4-nitro-L-phenylalanine (450%). Homology modeling and docking simulations were carried out and were consistent with the observed substrate specificity. To demonstrate the synthetic potential of AADC-BA, we carried out the production of serotonin by decarboxylation of L-HTP. The reaction yield of serotonin reached 98% after 1 h at the reaction conditions of 50 mM L-HTP and 4 U/mL AADC-BA. Moreover, we carried out preparative-scale decarboxylation of L-Phe (100 mM in 40-mL reaction mixture) and isolated the resulting 2-phenylethylamine (51% recovery yield). We expect that the broad substrate specificity of AADC-BA can be exploited to produce various aromatic biogenic amines. KEY POINTS: ⢠AADC-BA showed broad substrate specificity for various aromatic amino acids. ⢠The substrate specificity was elucidated by in silico structural modeling. ⢠The synthetic potential of AADC-BA was demonstrated for the production of biogenic amines.
Assuntos
Descarboxilases de Aminoácido-L-Aromático , Bacillus , 5-Hidroxitriptofano , Descarboxilases de Aminoácido-L-Aromático/genética , Serotonina , TriptofanoRESUMO
5-Acetoxymethyl-2-furfural (AMF) was prepared from D-fructose via 1,6-diacetylfructose (DAF) through a simple two-step reaction pathway. Immobilized enzyme (Novozym 435) was found to be the best enzymatic catalyst for the trans-esterification step (yielding 94.6% DAF). In the dehydration step, while soluble H2SO4 was found to be the best acidic catalyst (yielding 86.6% AMF), we opted to utilize heterogeneous cation exchange resin (Amberlyst 15) together with recyclable industrial solvents (1,4-dioxane) for a more sustainable AMF synthesis procedure. Although the total yield of AMF was a little lower, both the enzyme and the solid acid catalyst could be recycled for five cycles without a significant loss of activity, which has a major contribution to the cost-efficient aspect of the entire process.
Assuntos
Resinas de Troca de Cátion/química , Enzimas Imobilizadas , Frutose/química , Furaldeído/química , Lipase/química , Catálise , Desidratação , Esterificação , Solventes/químicaRESUMO
ω-Transaminase (ω-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most ω-TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective ω-TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for α-keto acids and α-amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid and L-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an α-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure L- and D-norvaline (i.e., enantiomeric excess [ee]>99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively.
Assuntos
Aminoácidos/biossíntese , Ochrobactrum anthropi/enzimologia , Engenharia de Proteínas , Transaminases/genética , Transaminases/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Simulação de Acoplamento Molecular , Mutagênese Sítio-DirigidaRESUMO
ω-Transaminase (ω-TA) is one of the important biocatalytic toolkits owing to its unique enzyme property which enables the transfer of an amino group between primary amines and carbonyl compounds. In addition to preparation of chiral amines, ω-TA reactions have been exploited for the asymmetric synthesis of L-amino acids using (S)-selective ω-TAs. However, despite the availability of (R)-selective ω-TAs, catalytic utility of the ω-TAs has not been explored for the production of D-amino acids. Here, we investigated the substrate specificity of (R)-selective ω-TAs from Aspergillus terreus and Aspergillus fumigatus and demonstrated the asymmetric synthesis of D-amino acids from α-keto acids. Substrate specificity toward D-amino acids and α-keto acids revealed that the two (R)-selective ω-TAs possess strict steric constraints in the small binding pocket that precludes the entry of a substituent larger than an ethyl group, which is reminiscent of (S)-selective ω-TAs. Molecular models of the active site bound to an external aldimine were constructed and used to explain the observed substrate specificity and stereoselectivity. α-Methylbenzylamine (α-MBA) showed the highest amino donor reactivity among five primary amines (benzylamine, α-MBA, α-ethylbenzylamine, 1-aminoindan, and isopropylamine), leading us to employ α-MBA as an amino donor for the amination of 5 reactive α-keto acids (pyruvate, 2-oxobutyrate, fluoropyruvate, hydroxypyruvate, and 2-oxopentanoate) among 17 ones tested. Unlike the previously characterized (S)-selective ω-TAs, the enzyme activity of the (R)-selective ω-TAs was not inhibited by acetophenone (i.e., a deamination product of α-MBA). Using racemic α-MBA as an amino donor, five D-amino acids (D-alanine, D-homoalanine, D-fluoroalanine, D-serine, and D-norvaline) were synthesized with excellent product enantiopurity (enantiomeric excess >99.7 %).
Assuntos
Aminoácidos/metabolismo , Aspergillus/enzimologia , Domínio Catalítico , Transaminases/química , Transaminases/metabolismo , Cetoácidos/metabolismo , Modelos Moleculares , Especificidade por SubstratoRESUMO
Removal of a side product to overcome unfavorable equilibrium is a prerequisite for the asymmetric amination of ketones using ω-transaminase (ω-TA). Alanine has been preferred as an amino donor because its deamination product (i.e. pyruvate) is easily removable by several enzymatic methods. Here, we demonstrated that the removal of pyruvate by an innate metabolic pathway could afford equilibrium shift of the ω-TA reactions.
Assuntos
Alanina/metabolismo , Cetonas/metabolismo , Transaminases/metabolismo , Aminação , Ácido Pirúvico/metabolismoRESUMO
Repurposing the intrinsic properties of natural enzymes can offer a viable solution to current synthetic challenges through the development of novel biocatalytic processes. Although amino acid racemases are ubiquitous in living organisms, an amine racemase (AR) has not yet been discovered despite its synthetic potential for producing chiral amines. Here, we report the creation of an AR based on the serendipitous discovery that amine transaminases (ATAs) can perform stereoinversion of 2-aminobutane. Kinetic modeling revealed that the unexpected off-pathway activity results from stereochemically promiscuous futile cycles due to incomplete stereoselectivity for 2-aminobutane. This finding motivated us to engineer an S-selective ATA through in silico alanine scanning and empirical combinatorial mutations, creating an AR with broad substrate specificity. The resulting AR, carrying double point mutations, enables the racemization of both enantiomers of diverse chiral amines in the presence of a cognate ketone. This strategy may be generally applicable to a wide range of transaminases, paving the way for the development of new-to-nature racemases.
Assuntos
Aminas , Racemases e Epimerases , Aminas/química , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Ciclização de Substratos , Biocatálise , Transaminases/metabolismo , Especificidade por Substrato , EstereoisomerismoRESUMO
ω-Transaminases display complicated inhibitions by ketone products and both enantiomers of amine substrates. Here, we report the first example of ω-transaminase devoid of such inhibitions. Owing to the lack of enzyme inhibitions, the ω-transaminase from Ochrobactrum anthropi enabled efficient kinetic resolution of α-methylbenzylamine (500 mM) even without product removal.
Assuntos
Inibidores Enzimáticos/metabolismo , Ochrobactrum anthropi/enzimologia , Fenetilaminas/metabolismo , Transaminases/metabolismo , Cinética , Estrutura Molecular , Transaminases/químicaRESUMO
Isopropylamine is an ideal amino donor for reductive amination of carbonyl compounds by ω-transaminase (ω-TA) owing to its cheapness and high volatility of a ketone product. Here we developed asymmetric synthesis of unnatural amino acids via ω-TA-catalyzed amino group transfer between α-keto acids and isopropylamine.
Assuntos
Aminoácidos/biossíntese , Propilaminas/metabolismo , Transaminases/metabolismo , Aminoácidos/química , Biocatálise , Estrutura Molecular , Propilaminas/química , Transaminases/químicaRESUMO
Secondary structure matters: We have constructed artificial intragenic riboswitches to probe ribosome accessibility to the 5' mRNA coding region at three-base resolution in Escherichia coli. We show that only mRNA folding stability in the +1 to +15 nt region affects the translation process.
Assuntos
Ligantes , RNA Mensageiro/metabolismo , Aptâmeros de Nucleotídeos/química , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , Dobramento de RNA , Estabilidade de RNA , RNA Mensageiro/química , Ribossomos/metabolismo , RiboswitchRESUMO
Chiral amines in enantiopure forms are important chemical building blocks, which are most well recognized in the pharmaceutical industries for imparting desirable biological activity to chemical entities. A number of synthetic strategies to produce chiral amines via biocatalytic as well as chemical transformation have been developed. Recently, ω-transaminase (ω-TA) has attracted growing attention as a promising catalyst which provides an environment-friendly access to production of chiral amines with exquisite stereoselectivity and excellent catalytic turnover. To obtain enantiopure amines using ω-TAs, either kinetic resolution of racemic amines or asymmetric amination of achiral ketones is employed. The latter is usually preferred because of twofold higher yield and no requirement of conversion of a ketone product back to racemic amine. However, the choice of a production process depends on several factors such as reaction equilibrium, substrate reactivity, enzyme inhibition, and commercial availability of substrates. This review summarizes the biochemical features of ω-TA, including reaction chemistry, substrate specificity, and active site structure, and then introduces recent advances in expanding the scope of ω-TA reaction by protein engineering and public database searching. We also address crucial factors to be considered for the development of efficient ω-TA processes.
Assuntos
Aminas Biogênicas/metabolismo , Biotecnologia/métodos , Tecnologia Farmacêutica/métodos , Transaminases/metabolismo , Domínio Catalítico , Modelos Químicos , Modelos Moleculares , Transaminases/químicaRESUMO
ω-Transaminase (ω-TA) is an industrially important enzyme for production of chiral amines. About 20 (S)-specific ω-TAs known to date show remarkably similar substrate selectivity characterized by stringent steric constraint precluding entry of a substituent larger than an ethyl group in the small binding pocket (S) and dual recognition of an aromatic substituent as well as a carboxylate group in the large pocket (L). The strictly defined substrate selectivity of the available ω-TAs remains a limiting factor in the production of structurally diverse chiral amines. In this work, we cloned, purified, and characterized three new ω-TAs from Ochrobactrum anthropi, Acinetobacter baumannii, and Acetobacter pasteurianus that were identified by a BLASTP search using the previously studied ω-TA from Paracoccus denitrificans. All the new ω-TAs exhibited similar substrate specificity, which led us to explore whether the molecular determinants for the substrate specificity are conserved among the ω-TAs. To this end, key active site residues were identified by docking simulation using the X-ray structure of the ω-TA from Pseudomonas putida. We found that the dual recognition in the L pocket is ascribed to Tyr23, Phe88*, and Tyr152 for hydrophobic interaction and Arg414 for recognition of a carboxylate group. In addition, the docking simulation indicates that Trp60 and Ile262 form the S pocket where the substituent size up to an ethyl group turns out to be sterically allowed. The six key residues were found to be essentially conserved among nine ω-TA sequences, underlying the molecular basis for the high similarity in the substrate selectivity.
Assuntos
Acinetobacter/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ochrobactrum anthropi/enzimologia , Transaminases/química , Acinetobacter/química , Acinetobacter/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ochrobactrum anthropi/química , Ochrobactrum anthropi/genética , Paracoccus denitrificans/química , Paracoccus denitrificans/enzimologia , Alinhamento de Sequência , Especificidade por Substrato , Transaminases/genética , Transaminases/metabolismoRESUMO
Porous TiO(2) nanotube arrays with three-dimensional (3D) interconnectivity were prepared using a sol-gel process assisted by poly(vinyl chloride-graft-4-vinyl pyridine), PVC-g-P4VP graft copolymer and a ZnO nanorod template. A 7 µm long ZnO nanorod array was grown from the fluorine-doped tin oxide (FTO) glass via a liquid phase deposition method. The TiO(2) sol-gel solution templated by the PVC-g-P4VP graft copolymer produced a random 3D interconnection between the adjacent ZnO nanorods during spin coating. Upon etching of ZnO, TiO(2) nanotubes consisting of 10-15 nm nanoparticles were generated, as confirmed by wide-angle x-ray scattering (WAXS), energy-filtering transmission electron microscopy (EF-TEM) and field-emission scanning electron microscopy (FE-SEM). The ordered and interconnected nanotube architecture showed an enhanced light scattering effect and increased penetration of polymer electrolytes in dye-sensitized solar cells (DSSC). The energy conversion efficiency reached 1.82% for liquid electrolyte, and 1.46% for low molecular weight (M(w)) and 0.74% for high M(w) polymer electrolytes.
Assuntos
Corantes/química , Nanotubos/química , Polivinil/síntese química , Energia Solar , Titânio/química , Cloreto de Vinil/síntese química , Eletrólitos/química , Nanotubos/ultraestrutura , Polimerização , Polivinil/química , Porosidade , Cloreto de Vinil/química , Difração de Raios X , Óxido de Zinco/químicaRESUMO
Genetic regulation by noncoding RNA elements such as microRNA and small interfering RNA (siRNA) involves hybridization of a short single-stranded RNA with a complementary segment in a target mRNA. The physical basis of the hybridization process between the structured nucleic acids is not well understood primarily because of the lack of information about the transition-state structure. Here we use transition-state theory, inspired by phi-value analysis in protein folding studies, to provide quantitative analysis of the relationship between changes in the secondary structure stability and the activation free energy. Time course monitoring of the hybridization reaction was performed under pseudo-steady-state conditions using a single fluorophore. The phi-value analysis indicates that the native secondary structure remains intact in the transition state. The nativelike transition state was confirmed via examination of the salt dependence of the hybridization kinetics, indicating that the number of sodium ions associated with the transition state was not substantially affected by changes in the native secondary structure. These results propose that hybridization between structured nucleic acids undergoes a transition state leading to formation of a nucleation complex and then is followed by sequential displacement of preexisting base pairings involving successive small energy barriers. The proposed mechanism might provide new insight into physical processes during small RNA-mediated gene silencing, which is essential to selection of a target mRNA segment for siRNA design.
Assuntos
DNA/química , Hibridização de Ácido Nucleico , Sequência de Bases , Calorimetria , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica , Cinética , MicroRNAs/química , MicroRNAs/genética , Modelos Teóricos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/isolamento & purificação , TermodinâmicaRESUMO
We have demonstrated the self-assembled DNA nanoparticles capable of controlled disassembly in response to a single nucleotide change (SNC) in a target nucleic acid. The DNA nanoparticles (avg diameter=51+/-22 nm) were constructed by joining two types of streptavidin-DNA conjugates with 2 molar equiv of a linker strand that carries complementary sequences to both conjugates. Nanoparticle disassembly triggered by a target strand (i.e., a perfect complement to the linker) selectively over mismatched targets was achieved by kinetically controlled nucleation occurring at a 6-nt overhang in the linker. The disassembly process was shown to be dramatically slowed down when using mismatch targets in which the SNC was positioned at the fourth nucleotide from the 3'-end. To verify whether the controlled disassembly also works for a SNC located in the middle of a target strand, we tested a deleterious Z variant (G1024A) of human alpha(1)-antitrypsin as a mismatch target (60-nt) carrying the point mutation at position 39. The wild-type target completed the disassembly process in less than 10 min, whereas the mismatch Z-type target could not complete the disassembly even in 3 h. The DNA nanoparticles are promising for sequence-dependent controlled release of short nucleic acids, including siRNA and antisense oligonucleotides, and construction of smart nanomaterials capable of sensing and processing single-nucleotide polymorphisms.
Assuntos
Pareamento Incorreto de Bases , DNA/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Nanopartículas/uso terapêutico , Oligonucleotídeos/administração & dosagem , Reagentes de Ligações Cruzadas , DNA/química , DNA/genética , Humanos , Cinética , Ácidos Nucleicos/química , Oligonucleotídeos/genética , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Estreptavidina , alfa 1-Antitripsina/genéticaRESUMO
Reliable kinetic parameters of enzymes are of paramount importance for a precise understanding of catalytic performance, which is essential for enzyme engineering and process optimization. Here, we developed a simple and convenient method to determine intrinsic kinetic parameters of R-selective ω-transaminases (ω-TAs) with a minimal set of kinetic data. Using (R)-α-methylbenzylamine ((R)-α-MBA) and pyruvate as a substrate pair, two R-selective ω-TAs from Arthrobacter sp. and Aspergillus fumigatus were subjected to kinetic measurements. In contrast to S-selective ω-TAs, both R-selective ω-TAs were observed to be devoid of substrate inhibition by pyruvate. Double reciprocal plot analysis was carried out with two sets of kinetic data obtained at varying concentrations of (R)-α-MBA under a fixed concentration of pyruvate and vice versa, leading to the determination of three intrinsic kinetic parameters, i.e., one kcat and two KM values, using three regression constants. The validity of the kinetic parameters was verified by a self-consistency test using a regression constant left out in the kinetic parameter determination, showing that deviations of calculated regression constants from the experimental ones were less than 15%. Because the kinetic parameters for (R)-α-MBA and pyruvate are not apparent but intrinsic, a cosubstrate substitution method enabled rapid determination of intrinsic parameters for a new substrate pair using just one set of kinetic data. Eventually, computational modeling of kinetic resolution of rac-α-MBA was carried out and showed a good agreement with experimental reaction progresses.
Assuntos
Arthrobacter/enzimologia , Aspergillus fumigatus/enzimologia , Proteínas de Bactérias/química , Proteínas Fúngicas/química , Fenetilaminas/química , Transaminases/química , Cinética , Ácido Pirúvico/químicaRESUMO
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol + acetate + pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol + acetate + pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol + acetate + pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1-2.3 mol(ethanol + acetate + pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.
RESUMO
Nucleic acid hybridization is one of the essential biological processes involved in storage and transmission of genetic information. Here we quantitatively determined the effect of secondary structure on the hybridization activation energy using structurally defined oligonucleotides. It turned out that activation energy is linearly proportional to the length of a single-stranded region flanking a nucleation site, generating a 0.18 kcal/mol energy barrier per nucleotide. Based on this result, we propose that the presence of single-stranded segments available for non-productive base pairing with a nucleation counterpart extends the searching process for nucleation sites to find a perfect match. This result may provide insights into rational selection of a target mRNA site for siRNA and antisense gene silencing.
Assuntos
DNA/química , Hibridização Genética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodos , Sequência de Bases , Ácidos Carboxílicos/química , Corantes Fluorescentes/química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genéticaRESUMO
Bacterial sensors, based on ligand-mediated genetic control systems, are promising for on-site chemical detection because sensing targets and generating signals do not require costly instrumentation. Here, we have constructed intragenic synthetic riboswitches without relying on high-throughput screening and demonstrated that the riboswitches can be harnessed to develop bacterial sensors displaying readily visible reporter signals in response to theophylline. In vivo imaging using the riboswitch showed target-specific changes in the expression of a green fluorescence protein reporter, which was visible even to the naked eye.
Assuntos
Técnicas Biossensoriais/métodos , Regulação Bacteriana da Expressão Gênica , Teofilina/isolamento & purificação , Sequência de Bases , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência MolecularRESUMO
Asymmetric synthesis of an unnatural amino acid was demonstrated by omega-transaminase from Vibrio fluvialis JS17. L-2-Aminobutyric acid was synthesized from 2-oxobutyric acid and benzylamine with an enantiomeric excess higher than 99%. The reaction showed severe product inhibition by benzaldehyde, which was overcome by employing a biphasic reaction system to remove the inhibitory product from the aqueous phase. In a typical biphasic reaction (50 mM 2-oxobutyric acid, 70 mM benzylamine and 2.64 U/ml purified enzyme) using hexane as an extractant, conversion of 2-oxobutyric acid reached 96% in 5 h whereas only 39% conversion was obtained without the product extraction.