RESUMEN
Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of l-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward l-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55-60 °C and pH 9.0-9.5. However, four of them, LsPAL1-LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.
Asunto(s)
Aminoácidos , Fenilanina Amoníaco-Liasa , Fenilanina Amoníaco-Liasa/genética , Lactuca/genética , Amoníaco , FenilalaninaRESUMEN
Herein, a Zr-based dual-ligand MOFs with pre-installed Rh complex was employed for NADH regeneration in situ and also used for immobilization of formic acid dehydrogenase (FDH) in order to realize a highly efficient CO2 fixation system. Then, based on the detailed investigations into the photochemical and electrochemical properties, it is demonstrated that the introduction of the photosensitive meso-tetra(4-carboxyphenyl) porphin (TCPP) ligands increased the catalytic active sites and improved photoelectric properties. Furthermore, the electron mediator Rh complex, anchored on the zirconium-based dual-ligand MOFs, enhanced the efficiency of electron transfer efficiency and facilitated the separation of photogenerated electrons and holes. Compared with UiO-66-NH2 , Rh-H2 TCPP-UiO-66-NH2 exhibits an optimized valence band structure and significantly improved photocatalytic activity for NAD+ reduction, resulting the synthesis of formic acid from CO2 increased from 150â µg mL-1 (UiO-66-NH2 ) to 254â µg mL-1 (Rh-H2 TCPP-UiO-66-NH2 ). Moreover, the assembled photocatalyst-enzyme coupled system also allows facile recycling of expensive electron mediator, enzyme, and photocatalyst.
RESUMEN
Following a synthetic chemistry blueprint for the valorization of lignocellulosic platform chemicals, this study showcases a so far unprecedented approach to implement non-natural enzyme modules inâ vivo. For the design of a novel functional whole cell tool, two purely abiotic transformations, a styrene monooxygenase-catalyzed Achmatowicz rearrangement and an alcohol dehydrogenase-mediated borrowing hydrogen redox isomerization, were incorporated into a recombinant bacterial host. Introducing this type of chemistry otherwise unknown in biosynthesis, the cellular factories were enabled to produce complex lactone building blocks in good yield from bio-based furan substrates. This whole cell system streamlined the synthetic cascade, eliminated isolation and purification steps, and provided a high degree of stereoselectivity that has so far been elusive in the chemical methodology.
Asunto(s)
Alcohol Deshidrogenasa , Furanos , Oxidación-Reducción , Lactonas , BiocatálisisRESUMEN
The addition of water to alkenes is an important method for the synthesis of alcohols, but the regioselectivity of acid-catalyzed hydration of terminal alkenes yields secondary alcohols according to Markovnikov's rule, making it difficult to obtain primary alcohols. Here we report a styrene monooxygenase that catalyzes the anti-Markovnikov hydration of the terminal aryl alkenes under anaerobic conditions. This hydration provides primary alcohols in good yields (up to 100 %), excellent anti-Markovnikov regioselectivity (>99 : 1), and good enantiomeric purity (60-83 % ee). Residues Asn46, Asp100, and Asn309 are essential for catalysis suggesting an acid-base mechanism with a carbanion-like intermediate that could account for the anti-Markovnikov regioselectivity. Our work reveals a new enzymatic tool with unusual regioselectivity based on the promiscuous catalytic activity of a monooxygenase.
Asunto(s)
Alcoholes , Alquenos , Alcoholes/química , Alquenos/química , Catálisis , EstereoisomerismoRESUMEN
Protein stability is crucial in enzymatic catalysis. To improve the efficiency in the searching for thermostablizing mutations, we applied a sequence consensus approach focusing on dimeric interface residues of ketoreductase ChKRED20. The strategy returned a success rate of 43%, revealing 9 beneficial mutations from 21 candidates with improved kinetic or thermodynamic stability. Several combinatorial mutants were then constructed, and mutant M8K displayed the highest thermostability, with a melting temperature (Tm) of 89 °C and a half-inactivation temperature (T50) of 93.4 °C, both of over 35 °C increase compared to the wild-type. M8K could remain stable for at least 7 days at its optimal reaction temperature of 55 °C. Its inactivation half-life (t1/2) was 110 min at 90 °C, while the wild-type was 18.6 min at 60 °C. The results were interpreted in the context of structural and molecular dynamic simulation analysis, which revealed the addition of intramolecular interactions, decreased conformational flexibility and increased compactness, all in agreement with the observed effect.
Asunto(s)
Estabilidad de Enzimas , Consenso , Cinética , Mutagénesis , Mutagénesis Sitio-Dirigida , TemperaturaRESUMEN
Asymmetric epoxidation catalyzed with styrene monooxygenase (SMO) is a powerful enzymatic process producing enantiopure styrene epoxide derivatives. To establish a more diversified reservoir of SMOs, a new SMO from Bradyrhizobium sp. ORS 375, named BrSMO, was mined from the database and characterized. BrSMO was constituted of an epoxygenase component of 415 amino acid residues and an NADH-dependent flavin reductase component of 175 residues. BrSMO catalyzed the epoxidation of styrene and 7 more styrene derivatives, yielding the corresponding (S)-epoxides with excellent enantiomeric excesses (95- > 99% ee), with the highest activity achieved for styrene. BrSMO also catalyzed the asymmetric sulfoxidation of 7 sulfides, producing the corresponding (R)-sulfoxides (20-90% ee) with good yields.
Asunto(s)
Proteínas Bacterianas/química , Bradyrhizobium/enzimología , Oxigenasas/química , Sulfóxidos/síntesis química , Catálisis , Sulfóxidos/químicaRESUMEN
Styrene monooxygenases (SMOs) are two-component enzymes known to catalyze the epoxidation of styrene to (S)-styrene oxide. In this work, we identified a new oxygenase component, named StStyA, from the genome of Streptomyces sp. NRRL S-31. StStyA displayed complementary stereoselectivity to all of the known SMOs when coupled with a known reductase component (PsStyB), which made it the first natural SMO that produces (R)-styrene oxide. Accordingly, a plasmid co-expressing StStyA and PsStyB was constructed, which led to an artificial two-component SMO, named StStyA/B. When applied in the bio-epoxidation of nine aromatic alkenes, the enzyme showed activity toward five alkenes, and consistently displayed (R)-selectivity. Excellent stereoselectivity was achieved for all five substrates with enantiomeric excesses ranging from 91% to >99%ee.
Asunto(s)
Proteínas Bacterianas/metabolismo , Oxigenasas/metabolismo , Streptomyces/enzimología , Proteínas Bacterianas/genética , Biocatálisis , Compuestos Epoxi/metabolismo , Cinética , Oxigenasas/genética , Streptomyces/genéticaRESUMEN
ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD+ complex at a resolution of 1.6 Å, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with kcat/Km increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with >90% isolated yield and 97 - >99%ee.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Chryseobacterium/enzimología , Cetonas/metabolismo , Oxidorreductasas de Alcohol/genética , Alcoholes/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Biocatálisis , Chryseobacterium/genética , Cristalografía por Rayos X , Mutación con Ganancia de Función , Cetonas/química , Cinética , Simulación del Acoplamiento Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Relación Estructura-ActividadRESUMEN
Ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate((S)-HEES)acts as a key chiral intermediate for the blockbuster antidepressant drug duloxetine, which canbe achieved viathe stereoselective bioreduction ofethyl 3-oxo-3-(2-thienyl) propanoate (KEES) that containsa 3-oxoacyl structure.The sequences of the short-chain dehydrogenase/reductases from Chryseobacterium sp. CA49 were analyzed, and the putative3-oxoacyl-acyl-carrier-protein reductase, ChKRED12, was able to stereoselectivelycatalyze theNADPH-dependent reduction to produce (S)-HEES.The reductase activity of ChKRED12 towardsothersubstrates with 3-oxoacyl structure were confirmed with excellent stereoselectivity (>99% enantiomeric excess) in most cases. When coupled with a cofactor recycling system using glucose dehydrogenase, the ChKRED12 was able to catalyze the complete conversion of 100 g/l KEES within 12h, yielding the enantiopure product with >99% ee, showing a remarkable potential to produce (S)-HEES.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/metabolismo , Proteínas Bacterianas/metabolismo , Propionatos/metabolismo , Deshidrogenasas-Reductasas de Cadena Corta/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catálisis , Chryseobacterium/enzimología , Chryseobacterium/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa 1-Deshidrogenasa/metabolismo , Cinética , Oxidación-Reducción , Propionatos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deshidrogenasas-Reductasas de Cadena Corta/química , Deshidrogenasas-Reductasas de Cadena Corta/genética , Estereoisomerismo , Especificidad por SustratoRESUMEN
Styrene monooxygenases (SMOs) are highly stereoselective enzymes that catalyze the formation of chiral epoxides as versatile building blocks. To expand the enzyme toolbox, two bacterial SMOs were identified from the genome of marine microbes Paraglaciecola agarilytica NO2 and Marinobacterium litorale DSM 23545, and heterologously expressed in Escherichia coli in soluble form. Both of the resulting whole-cell biocatalysts exhibited maximal activity at 30⯰C and pH 8.0. They catalyzed the sulfoxidation reactions, and the epoxidation of both conjugated and unconjugated styrene derivatives with up to >99%ee. MlSMO displayed higher activity toward most substrates tested. Compared to an established SMO from Pseudomonas species (PsSMO), MlSMO achieved 3.0-, 3.4- and 2.6-fold conversions for substrates styrene, cinnamyl alcohol and 4-vinyl-2, 3-dihydrobenzofuran, respectively.
Asunto(s)
Alteromonadaceae/enzimología , Proteínas Bacterianas/metabolismo , Oceanospirillaceae/enzimología , Oxigenasas/metabolismo , Alquenos/química , Alquenos/metabolismo , Alteromonadaceae/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Biocatálisis , Biotransformación , Concentración de Iones de Hidrógeno , Cinética , Oceanospirillaceae/genética , Oxigenasas/genética , Pseudomonas/enzimología , Pseudomonas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato , TemperaturaRESUMEN
ChKRED20 is an efficient and robust anti-Prelog ketoreductase that can catalyze the reduction of ketones to chiral alcohols as pharmaceutical intermediates with great industrial potential. To overcome its limitation on the bioreduction of ortho-substituted acetophenone derivatives, the X-ray crystal structure of the apo-enzyme of ChKRED20 was determined at a resolution of 1.85 Å and applied to the molecular modeling and reshaping of the catalytic cavity via three rounds of iterative saturation mutagenesis together with alanine scanning and recombination. The mutant Mut3B was achieved with expanded catalytic scope that covered all the nine substrates tested as compared with two substrates for the wild type. It exhibited 13-20-fold elevated k cat/K m values relative to the wild type or to the first gain-of-activity mutant, while retaining excellent stereoselectivity toward seven of the substrates (98-> 99% ee). Another mutant 29G10 displayed complementary selectivity for eight of the ortho-substituted acetophenone derivatives, with six of them delivering excellent stereoselectivity (90-99% ee). Its k cat/K m value toward 1-(2-fluorophenyl)ethanone was 5.6-fold of the wild type. The application of Mut3B in elevated substrate concentrations of 50-100 g/l was demonstrated in 50-ml reactions, achieving 75-> 99% conversion and > 99% ee.
Asunto(s)
Chryseobacterium/enzimología , Cetonas/metabolismo , Mutagénesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Oxidorreductasas/química , Conformación Proteica , Especificidad por SustratoRESUMEN
Cytochrome P450 enzymes are versatile biocatalysts with great potential in biotechnology. A new bacterial P450 was identified from the genome of Rhodococcus wratislaviensis NBRC 100605 and annotated as CYP108N7. The enzyme accepted the ferredoxin and ferredoxin reductase from spinach as surrogate redox partners for improved electron transfer efficiency. It was heterologous expressed in Escherichia coli together with the redox partners and a glucose dehydrogenase which supplied the reduced cofactor NADPH. The resulting whole-cell biocatalyst catalyzed a variety of reactions including sulfoxidation, epoxidation, hydroxylation, demethylation and dehalogenation. Remarkable stereoselectivity was observed in asymmetric sulfoxidation reaction, which could deliver chiral sulfoxides with >99% ee from thioanisole and derivatives.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/química , Rhodococcus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Biocatálisis , Biotecnología , Biotransformación , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Glucosa 1-Deshidrogenasa/genética , Glucosa 1-Deshidrogenasa/metabolismo , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/genética , Homología de Secuencia de Aminoácido , Espectrofotometría , Especificidad por SustratoRESUMEN
Radix Sanguisorbae, the root of Sanguisorba officinalis L. is used as traditional Chinese medicine. In recent decades, it has been reported to be clinically effective against myelosuppression induced by chemotherapy and/ or radiotherapy. However, the underlining mechanism has not been well studied. In this work, we evaluated the hematopoietic effect of total saponins from S. officinalis L. on myelosuppressive mice induced by cyclophosphamide and by60Co-γ-irradiation and confirmed the therapeutic effect. Then, we found total saponins and their characteristic constituents Ziyuglycoside I and Ziyuglycoside II can inhibit apoptosis of TF-1 cells caused by cytokine deprivation, and promote survival of mouse bone marrow nuclear cells through focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (Erk1/2) activation in vitro. In addition, they can down-regulate macrophage inflammatory protein 2 (MIP-2), platelet factor 4 (PF4) and P-selectin secretion, which are reported to be suppressive to hematopoiesis, both in vitro and in vivo. These results suggest that promotion of survival through FAK and Erk1/2 activation and inhibition of suppressive cytokines in the bone marrow is likely to be the pharmacological mechanism underlying the hematopoietic effect of saponins from S. officinalis L.
RESUMEN
The synthesis of optically pure secondary epoxy alcohols from racemic allylic alcohols using a single whole-cell biocatalyst of recombinant Escherichia coli coexpressing three oxidoreductases is described. The cascade involves the concurrent action of a styrene monooxygenase that catalyzes the formation of the chiral epoxy group, and two alcohol dehydrogenases that fulfil the epimerisation of the hydroxy group. Two sets of alcohol dehydrogenases were each applied to couple with styrene monooxygenase in order to realize the epimerisation in a stereo-complementary manner. Excellent enantio- and diastereo-selectivities were achieved for most of the 12 substrates.
RESUMEN
(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity. Then, the two critical positions were each randomized by constructing saturation mutagenesis libraries, which delivered several mutants with further enhanced activity. Among them, the mutant L205A was the best performer with a specific activity of 178 µmol/min/mg, ten times of that of the wild-type. Its k cat/K m increased by 15 times and half-life at 50 °C increased by 70 %. The mutant catalyzed the complete conversion of 150 and 200 g/l substrate within 6 and 20 h, respectively, to yield enantiopure (S)-CFPL with an isolated yield of 95 %.
Asunto(s)
Adenosina/análogos & derivados , Chryseobacterium/enzimología , Etanol/análogos & derivados , Etanol/síntesis química , Cetonas/metabolismo , Oxidorreductasas/metabolismo , 2-Propanol/química , Adenosina/síntesis química , Adenosina/química , Biocatálisis , Chryseobacterium/metabolismo , Etanol/química , Biblioteca de Genes , Mutagénesis , NAD/química , Oxidación-Reducción , Oxidorreductasas/genética , Especificidad por Sustrato , TicagrelorRESUMEN
Efficient asymmetric bio-epoxidation of electron-deficient α,ß-unsaturated ketones was realized via a tandem reduction-epoxidation-dehydrogenation cascade, which proceeds in a switchable manner to afford either chiral epoxy ketones or allylic epoxy alcohols with up to >99% yield and >99%ee.
Asunto(s)
Compuestos Epoxi/química , Cetonas/químicaRESUMEN
Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate for the synthesis of "blockbuster" drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bio-reductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of ChKRED20 to allow a higher reaction temperature. After one round of error-prone PCR (epPCR) library screening followed by the combination of beneficial mutations, a triple-mutant MC135 was successfully achieved with substantially enhanced thermostablity. The activity of MC135 at 50 °C was similar to the wild type. However, at its temperature optima of 65 °C, the mutant displayed 63 % increase of activity compared to the wild type and remained >95 % activity after being incubated for 15 days, while the wild type had a half-life of 11.9 min at 65 °C. At a substrate/catalyst ratio of 100 (w/w), the mutant catalyzed the complete conversion of 300 g/l substrate within 1 h to yield enantiopure (S)-CHBE with an isolated yield of 95 %, corresponding to a space-time yield of 1824 mM/h.
Asunto(s)
Acetoacetatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chryseobacterium/enzimología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Acetoacetatos/química , Biocatálisis , Chryseobacterium/química , Chryseobacterium/genética , Estabilidad de Enzimas , Calor , Isomerismo , Cinética , Mutación , Oxidorreductasas/químicaRESUMEN
Feruloyl esterase A from Aspergillus niger (AnFaeA) contains three intramolecular disulfide bonds and one free cysteine at position 235. Saturated mutagenesis at Cys235 was carried out to produce five active mutants, all of which displayed unusual thermal inactivation patterns with the most residual activity achieved at 75°C, much higher than the parental AnFaeA. But their optimal reaction temperatures were lower than the parental AnFaeA. Extensive investigation into their free thiol and disulfide bond, circular dichroism spectra and fluorescence spectra revealed that the unfolding of the parental enzyme was irreversible on all the tested conditions, while that of the Cys235 mutants was reversible, and their ability to refold was highly dependent on the denaturing temperature. Mutants denatured at 75°C were able to efficiently reverse the unfolding to regain native structure during the cooling process. This study provided valid evidence that free cysteine substitutions can reduce irreversible thermal inactivation of proteins.
Asunto(s)
Aspergillus niger/enzimología , Hidrolasas de Éster Carboxílico/química , Cisteína , Proteínas Fúngicas/química , Sustitución de Aminoácidos , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Dicroismo Circular , Activación Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pliegue de Proteína , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide enantiopure glycidol derivatives. To overcome the low theoretical yield of kinetic resolution, we designed a one-pot two-step enzymatic cascade using prochiral α,ß-unsaturated ketones as the substrates. An S-specific ketoreductase ChKRED03 was screened for the efficient bioreduction of the substrates to provide (S)-allylic alcohols, which underwent SMO-catalyzed epoxidation to achieve glycidol derivatives with contiguous stereogenic centers. Excellent enantioselectivity (ee > 99%) and diastereoselectivity (de > 99%) were achieved for the majority of the substrates, and product yields reached up to >99%.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Compuestos Epoxi/metabolismo , Oxigenasas/metabolismo , Propanoles/metabolismo , Biocatálisis , Compuestos Epoxi/química , Cinética , Propanoles/química , EstereoisomerismoRESUMEN
A putative enoate reductase, Achr-OYE4, was mined from the genome of Achromobacter sp. JA81, expressed in Escherichia coli, and was characterized. Sequence analysis and spectral properties indicated that Achr-OYE4 is a typical flavin mononucleotide-dependent protein; it preferred NADH over NADPH as a cofactor. The heterologously expressed protein displayed good activity and excellent stereoselectivity toward some activated alkenes in the presence of NADH, NADPH, or their recycling systems. The glucose dehydrogenase-based recycling system yielded the best results in most cases, with a product yield of up to 99 % and enantiopurity of >99 % ee. Achr-OYE4 is an important addition to the asymmetric reduction reservoir as an "old yellow enzyme" from Achromobacter.