RESUMEN
The Dietary Approaches to Stop Hypertension (DASH) diet is highly effective in controlling blood pressure (BP). Although Na restriction is not a primary focus within the DASH diet, it is recommended that it be added to control BP. Therefore, we aimed to systematically review the characteristics and BP-lowering effects of Na-restricted DASH diet interventions. We searched thirteen databases, namely, MEDLINE, Embase, Cochrane Central Register of Controlled Trials, KoreaMed, KISS, KMbase, RISS, CINAHL, Scopus, ClinicalTrials.gov, Grey Literature Report, OpenGrey and PQDT Global, for articles published through May 2023. The randomised controlled trials assessing the BP-lowering effect of the Na-restricted DASH diet in adults aged 18 years and older were included. The study protocol was registered in the PROSPERO registry (CRD42023409996). The risk of bias in the included studies was also assessed. Nine articles were included in this review. Interventions were categorised into three types: feeding, provision and education, and the study results were compared by intervention type. BP was significantly reduced in two of the three feeding studies, one of the three provisional studies and none of the educational studies. In eight studies, effect sizes varied among both systolic BP (-7·7 to -2·4) and diastolic BP (-8·3 to 0·1). Six studies showed an overall high risk of bias. In conclusion, Na-restricted DASH may have beneficial effects on BP control. Additionally, compared with control interventions, feeding interventions appeared to have a greater BP-lowering effect. Further high-quality studies are needed to improve the quality of the evidence.
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1,4-Butanediol (1,4-BDO) is a valuable industrial chemical that is primarily produced via several energy-intensive petrochemical processes based on fossil-based raw materials, leading to issues related to: non-renewability, environmental contamination, and high production costs. 1,4-BDO is used in many chemical reactions to develop a variety of useful, valuable products, such as: polyurethane, Spandex intermediates, and polyvinyl pyrrolidone (PVP), a water-soluble polymer with numerous personal care and pharmaceutical uses. In recent years, to satisfy the growing need for 1,4-BDO, there has been a major shift in focus to sustainable bioproduction via microorganisms using: recombinant strains, metabolic engineering, synthetic biology, enzyme engineering, bioinformatics, and artificial intelligence-guided algorithms. This article discusses the current status of the development of: various chemical and biological production techniques for 1,4-BDO, advances in biological pathways for 1,4-BDO biosynthesis, prospects for future production strategies, and the difficulties associated with environmentally friendly and bio-based commercial production strategies.
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Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.
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Proteínas de Escherichia coli/genética , Escherichia coli/genética , FMN Reductasa/genética , Regulación Bacteriana de la Expresión Génica , Indoles/metabolismo , Oxidorreductasas/genética , Oxigenasas/genética , Triptófano/metabolismo , Triptofanasa/genética , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Clonación Molecular , Colorantes/aislamiento & purificación , Colorantes/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , FMN Reductasa/metabolismo , Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Halogenación , Carmin de Índigo/aislamiento & purificación , Carmin de Índigo/metabolismo , Indoles/aislamiento & purificación , Ingeniería Metabólica/métodos , Oxidorreductasas/metabolismo , Oxigenasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semiconductores , Estereoisomerismo , Triptofanasa/metabolismoRESUMEN
6-Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin-derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site-specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6-halogenase fused with flavin reductase Fre (Fre-L3-SttH), tryptophanase (TnaA), toluene 4-monooxygenase (PmT4MO), and flavin-containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6-bromoindole with 45% yield to produce 6-bromo-2-oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen.
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Escherichia coli , Triptófano , Escherichia coli/metabolismo , Indoles , Oxígeno/metabolismo , Triptófano/metabolismoRESUMEN
PURPOSE: This study aimed to determine the relationships between occupational stress, sleep disturbance, and presenteeism in shiftwork nurses in South Korea. DESIGN: This cross-sectional study used survey data from 442 shiftwork nurses at two hospitals in South Korea. Data were collected between March 2018 and April 2020. METHODS: A self-reported questionnaire was used to assess shiftwork nurses' demographic and job-related information, presenteeism, occupational stress, and sleep disturbance. Data were analyzed using descriptive statistics, multiple regression, and the PROCESS macro. FINDINGS: The mean presenteeism score was 39.52. Multiple regression analysis revealed that job insecurity, interpersonal conflict, occupational climate, lack of reward, and job demand are significantly associated with presenteeism. Sleep disturbance was related to presenteeism and had a moderating effect on the relationship between occupational stress and presenteeism. CONCLUSIONS: This study indicates that proper occupational climate, positive interpersonal relationships, and adequate sleep can reduce shiftwork presenteeism. Therefore, individual and organizational efforts to alleviate occupational stress and sleep disturbances are essential. CLINICAL RELEVANCE: Organizational support is needed to maintain a positive occupational climate and interpersonal relationships and ameliorate sleep disturbance among shiftwork nurses.
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Enfermeras y Enfermeros , Estrés Laboral , Trastornos del Sueño-Vigilia , Estudios Transversales , Humanos , Estrés Laboral/epidemiología , Presentismo , Sueño , Trastornos del Sueño-Vigilia/epidemiología , Encuestas y CuestionariosRESUMEN
This study explored oncologists' experiences of delivering bad news to patients with cancer and their families. Nine oncologists recruited from three superior hospitals in Korea completed in-depth interviews. The results of thematic analyses identified four themes: precursors to bad news delivery, why it is difficult to deliver bad news, when it is more difficult to deliver bad news, and strategies of delivering bad news. The participants felt unprepared for the task and stressed because breaking bad news goes against their responsibility to do no harm and their professional objective to promote healing. Although they were unclear about best practices regarding communication styles, they individualized their communication style to meet the needs of their patients, who have an array of cultural, social, and spiritual backgrounds. Understanding oncologists' perceptions of bad news delivery can inform culturally appropriate interventions for alleviating their stress and improving patient-physician relationships in communication of bad news.
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Oncólogos , Revelación de la Verdad , Comunicación , Humanos , Relaciones Médico-Paciente , República de CoreaRESUMEN
Violacein is a pigment synthesized by Gram-negative bacteria such as Chromobacterium violaceum. It has garnered significant interest owing to its unique physiological and biological activities along with its synergistic effects with various antibiotics. In addition to C. violaceum, several microorganisms, including: Duganella sp., Pseudoalteromonas sp., Iodobacter sp., and Massilia sp., are known to produce violacein. Along with the identification of violacein-producing strains, the genetic regulation, quorum sensing mechanism, and sequence of the vio-operon involved in the biosynthesis of violacein have been elucidated. From an engineering perspective, the heterologous production of violacein using the genetically engineered Escherichia coli or Citrobacter freundii host has also been attempted. Genetic engineering of host cells involves the heterologous expression of genes involved in the vio operon and the optimization of metabolic pathways and gene regulation. Further, the crystallography of VioD and VioE was revealed, and mass production by enzyme engineering has been accelerated. In this review, we highlight the biologically assisted end-use applications of violacein (such as functional fabric development, nanoparticles, functional polymer composites, and sunscreen ingredients) and violacein activation mechanisms, production strains, and the results of mass production with engineered methods. The prospects for violacein research and engineering applications have also been discussed.
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Chromobacterium , Indoles , Chromobacterium/genética , Percepción de QuorumRESUMEN
Bacillus subtilis is regarded as a suitable host for biochemical production owing to its excellent growth and bioresource utilization characteristics. In addition, the distinct endogenous metabolic pathways and the suitability of the heterologous pathways have made B. subtilis a robust and promising host for producing biochemicals, such as: bioalcohols; bioorganic acids (lactic acids, α-ketoglutaric acid, and γ-aminobutyric acid); biopolymers (poly(γ-glutamic acid, polyhydroxyalkanoates (PHA), and polysaccharides and monosaccharides (N-acetylglucosamine, xylooligosaccharides, and hyaluronic acid)); and bioflocculants. Also for producing oligopeptides and functional peptides, owing to its efficient protein secretion system. Several metabolic and genetic engineering techniques, such as target gene overexpression and inactivation of bypass pathways, have led to the improvement in production titers and product selectivity. In this review article, recent progress in the utilization of robust B. subtilis-based host systems for biomass conversion and biochemical production has been highlighted, and the prospects of such host systems are suggested.
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Bacillus subtilis , Redes y Vías Metabólicas , Bacillus subtilis/genética , Biomasa , Ingeniería Genética , Ingeniería Metabólica , PéptidosRESUMEN
OBJECTIVE: The efficacy of antidepressants in post-stroke depressive symptoms (PSD) varies. We aimed to examine whether the effect of escitalopram on PSD differs according to individual depressive symptoms and stroke lesion location. METHODS: This is a post hoc analysis of EMOTION (ClinicalTrials.gov, NCT01278498), a randomized, placebo-controlled, double-blind trial that examined the efficacy of escitalopram on depression in acute stroke patients (237 with placebo, 241 with escitalopram). Depressive symptoms were evaluated with the 10-item Montgomery-Åsberg Depression Rating Scale (MADRS). Changes in MADRS and individual item scores at 12 weeks were compared between the treatment groups and among the stroke lesion location groups. Stroke lesion locations were grouped according to the anatomical distribution of serotonin fibers that originate from the midbrain/pons and spread to the forebrain via subcortical structures: "Midbrain-Pons," "Frontal-Subcortical," and "Others." Least-squares means were calculated to demonstrate the independent effect of lesion location. RESULTS: Total MADRS scores decreased more significantly in the escitalopram than in the placebo group, while a significant effect of escitalopram was observed in only 3 items: apparent sadness, reported sadness, pessimistic thoughts. In the lesion location analyses, escitalopram users in the Frontal-Subcortical group showed significant improvement in total MADRS scores (placebo [n = 130] vs. escitalopram [n = 148], least-square mean [95% CI]: -2.3 [-3.5 to -0.2] vs. -4.5 [-5.5 to -3.4], p = .005), while those in the Midbrain-Pons and Others groups did not. CONCLUSIONS: The effect of escitalopram on PSD may be more prominent in patients with particular depressive symptoms and stroke lesion locations, suggesting the need for tailored treatment strategies.
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Trastorno Depresivo Mayor , Accidente Cerebrovascular , Citalopram/uso terapéutico , Depresión/tratamiento farmacológico , Depresión/etiología , Método Doble Ciego , Escitalopram , Humanos , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Resultado del TratamientoRESUMEN
In this study, synthetic allomelanin was prepared from wild-type Streptomyces glaucescens and recombinant Escherichia coli BL21(DE3) strains. S. glaucescens could produce 125.25 ± 6.01 mg/L of melanin with a supply of 5 mM caffeic acid within 144 h. The ABTS radical scavenging capacity of S. glaucescens melanin was determined to be approximately 7.89 mg/mL of IC50 value, which was comparable to L-tyrosine-based eumelanin. The isolated melanin was used in cotton fabric dyeing, and the effect of copper ions, laccase enzyme treatment, and the dyeing cycle on dyeing performance was investigated. Interestingly, dyeing fastness was greatly improved upon treatment with the laccase enzyme during the cotton dyeing process. Besides, the supply of C5-diamine, which was reported to lead to more complex crosslinking between melanin units, to caffeic acid-based melanin synthesis was also investigated for higher production and novel functionalities. To facilitate the supply of caffeic acid and C5-diamine, E. coli strains expressing each or combinations of tyrosine ammonia lyase/p-coumarate 3-hydroxylase, feruloyl-CoA synthetase/enoyl-CoA hydratase/aldolase, and tyrosinase/lysine decarboxylase enzymes were prepared and investigated for their eumelanin, C5-diamine, and allomelanin production from L-tyrosine and L-lysine, respectively. Finally, H-NMR, FT-IR, and MALDI-TOF analysis of the synthetic melanin pigments were attempted to obtain the chemical information.
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Proteínas Bacterianas/metabolismo , Ácidos Cafeicos/química , Escherichia coli/metabolismo , Melaninas/metabolismo , Streptomyces/metabolismo , Tirosina/química , Antioxidantes/química , Escherichia coli/crecimiento & desarrollo , Streptomyces/crecimiento & desarrolloRESUMEN
Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l-tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.
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Proteínas Bacterianas/metabolismo , Oxidorreductasas/metabolismo , Streptomyces/enzimología , Triptófano/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Halogenación , Mutagénesis Sitio-Dirigida , Mutación , Oxidorreductasas/química , Oxidorreductasas/genética , Filogenia , Homología de Secuencia , Especificidad por Sustrato , Triptófano/químicaRESUMEN
BACKGROUND: Genetic variants may play a role in determining the location of cerebral atherosclerosis. We aimed to investigate the association between RNF213, MMP2, and genetic polymorphisms linked to vascular tortuosity with the location of cerebral arterial atherosclerosis. METHODS: A prospective case-control study was conducted on patients with ischemic stroke and age- and sex-matched stroke-free controls. The stroke patients were categorized into those with intracranial artery atherosclerosis (ICAS), extracranial artery atherosclerosis (ECAS), and small vessel occlusion (SVO). Six single nucleotide polymorphisms (SNPs) including rs2118181 (FBN1), rs2179357 (SLC2A10), rs1036095 (TGFBR2), rs243865 (MMP2), rs1800470 (TGFB1), and rs112735431 (RNF213) were analyzed with the TaqMan Genotyping Assay, and the distribution of genotypes across groups was compared. RESULTS: None of the 6 SNPs were associated with stroke on comparing the 449 stroke patients (71 with ECAS, 169 with ICAS, and 209 with SVO) to the 447 controls. In the subgroup analysis, the adjusted odds ratios (aORs) for age and sex indicated a significant association between rs112735431 and ICAS in the allele comparison analysis and in the additive and dominant model analyses. rs112735431 was associated with anterior circulation involvement and increased burden of cerebral atherosclerosis. rs2179357 was significantly associated with ICAS in the recessive model analysis, and rs1800470 was significantly associated with ECAS in the recessive model analysis when compared to controls. CONCLUSION: rs112735431 was associated with ICAS and increased atherosclerosis burden in Korean stroke patients. Further studies are needed to elucidate the role of rs112735431 and to confirm the association of rs2179357 and rs1800470 with cerebral atherosclerosis.
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Adenosina Trifosfatasas/genética , Enfermedades de los Pequeños Vasos Cerebrales/genética , Arteriosclerosis Intracraneal/genética , Polimorfismo de Nucleótido Simple , Accidente Cerebrovascular/genética , Ubiquitina-Proteína Ligasas/genética , Anciano , Estudios de Casos y Controles , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Femenino , Fibrilina-1/genética , Predisposición Genética a la Enfermedad , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Arteriosclerosis Intracraneal/diagnóstico por imagen , Masculino , Metaloproteinasa 2 de la Matriz/genética , Persona de Mediana Edad , Fenotipo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Medición de Riesgo , Factores de Riesgo , Seúl , Accidente Cerebrovascular/diagnóstico por imagen , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND: We aimed to examine sex differences in symptom characteristics and pharmacological responses in post-stroke depressive (PSD) symptoms. METHODS: This is a post hoc analysis of EMOTION (ClinicalTrials.gov, NCT01278498), a randomized, placebo-controlled, double-blind trial that examined the efficacy of escitalopram for 3 months on depression in patients with acute stroke. Depressive symptoms were evaluated using the 10-item Montgomery-Åsberg Depression Rating Scale (MADRS). Baseline characteristics, clinical variables, and treatment responses to escitalopram were compared between male and female patients. Treatment responses were defined as changes in MADRS (total score and its components) between baseline and 3 months and were compared between the escitalopram and placebo groups within each sex group. The least square mean was calculated to determine the independent effect of escitalopram, of which interaction was evaluated with patient sex. RESULTS: Of the 478 patients (intention-to-treat population), 187 (39%) were female. Female patients were significantly older than male patients and demonstrated more severe depressive symptoms at baseline (male vs. female, MADRS score, mean [SD]: 9.7 ± 8.0 vs. 12.2 ± 8.4, p = 0.001), especially in apparent sadness, reported sadness, and reduced appetite items. These differences were significant after adjustment for age and the severity of neurologic deficits. The female escitalopram group showed a significant 3-month improvement in MADRS scores (placebo [n = 86] vs. escitalopram [n = 101], least square mean [95% CI] -2.7 [-4.1 to -1.2] vs. -5.0 [-6.4 to -3.6], p = 0.007), and this efficacy was prominent in apparent sadness, reported sadness, and pessimistic thoughts items. However, there was no significant effect of escitalopram on depressive symptoms in the male group. The treatment responses of escitalopram tended to be more pronounced in the female group, particularly in alleviating a subset of depressive symptoms such as apparent sadness (p for interaction = 0.009). CONCLUSION: PSD may differ according to sex in its symptom characteristics and treatment responses to escitalopram, and tailored treatment strategies for PSD may therefore be needed.
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Afecto/efectos de los fármacos , Antidepresivos de Segunda Generación/uso terapéutico , Citalopram/uso terapéutico , Depresión/tratamiento farmacológico , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Accidente Cerebrovascular/complicaciones , Anciano , Anciano de 80 o más Años , Depresión/diagnóstico , Depresión/etiología , Depresión/psicología , Método Doble Ciego , Femenino , Disparidades en el Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , República de Corea , Factores de Riesgo , Factores Sexuales , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/psicología , Factores de Tiempo , Resultado del TratamientoRESUMEN
Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.
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Proteínas Bacterianas , Escherichia coli , Expresión Génica , Metiltransferasas , Microorganismos Modificados Genéticamente , Presión Osmótica/efectos de los fármacos , Cloruro de Sodio/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cupriavidus necator/enzimología , Cupriavidus necator/genética , Escherichia coli/enzimología , Escherichia coli/genética , Halomonas/enzimología , Halomonas/genética , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Microorganismos Modificados Genéticamente/enzimología , Microorganismos Modificados Genéticamente/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.
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Acetato CoA Ligasa/biosíntesis , Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Butanoles/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Ingeniería Metabólica/métodos , Acetato CoA Ligasa/genética , Escherichia coli/genéticaRESUMEN
In this study, protease Pph_Pro1 from Pseudoalteromonas phenolica, possessing extracellular proteolytic activity and salt tolerance, was investigated for cloning, expression, and purification purposes. Through optimization, it was determined that optimum soluble recombinant expression was achieved when Pph_Pro1 was co-expressed with the pTf16 vector chaperone in LB medium supplemented with CaCl2. Pph_Pro1 was purified using osmotic shock and immobilized metal-affinity chromatography (IMAC). Isolated Pph_Pro1 activity was measured as 0.44 U/mg using casein as a substrate. Interestingly, Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostable properties. Furthermore, it was resistant to several hydrophilic and hydrophobic organic solvents. Substrate specificity and kinetic values such as Km and Vmax were determined with casein, bovine serum albumin (BSA), and algal waste protein as substrates, indicating that the Pph_Pro1 protease enzyme had a greater affinity for casein. Based on the remarkable characteristics of this Pph_Pro1 protease enzyme, it can potentially be utilized in many biotechnological industries.
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Proteínas Bacterianas/genética , Escherichia coli/genética , Péptido Hidrolasas/genética , Pseudoalteromonas/enzimología , Proteínas Recombinantes de Fusión/genética , Proteínas Algáceas/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Cloruro de Calcio/farmacología , Caseínas/química , Cromatografía de Afinidad , Clonación Molecular , Medios de Cultivo/química , Medios de Cultivo/farmacología , Pruebas de Enzimas , Estabilidad de Enzimas , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/aislamiento & purificación , Proteolisis , Pseudoalteromonas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Salinidad , Tolerancia a la Sal/fisiología , Albúmina Sérica Bovina/química , Especificidad por SustratoRESUMEN
CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.
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Sistema Enzimático del Citocromo P-450/genética , Ácido Palmítico/metabolismo , Ingeniería de Proteínas/métodos , Catálisis , Dominio Catalítico , Colorimetría , Sistema Enzimático del Citocromo P-450/metabolismo , Bacteria Gordonia/enzimología , Bacteria Gordonia/metabolismo , Ensayos Analíticos de Alto Rendimiento , Hidroxilación , Cinética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Especificidad por SustratoRESUMEN
A potent phytoestrogen, (S)-equol, is a promising isoflavone derivative drawing our great attention owing to its various biological and clinical benefits. Through selective activation of the estrogen receptor ERß or androgen receptor, (S)-equol reduces menopausal symptoms, osteoporosis, skin aging, hair loss, and incidence of prostate or ovarian cancers without adverse effects. Traditional biosynthesis of (S)-equol exploited non-productive natural equol-producing anaerobic bacteria that mainly belong to Coriobacteriaceae isolated from human intestine. Recently, we developed a recombinant Escherichia coli strain which could convert daidzein into (S)-equol effectively under an aerobic condition. However, the yield was limited up to about the 200 mg/L level due to unknown reasons. In this study, we identified that the bottleneck of the limited production was the low solubility of isoflavone (i.e., 2.4 mg/L) in the reaction medium. In order to solve the solubility problem without harmful effect to the whole-cell catalyst, we applied commercial hydrophilic polymers (HPs) and a polar aprotic co-solvent in the reaction medium. Among the examined water-soluble polymers, polyvinylpyrrolidone (PVP)-40k was verified as the most promising supplement which increased daidzein solubility by 40 times and (S)-equol yield up to 1.22 g/L, the highest ever reported and the first g/L level biotransformation. Furthermore, PVP-40k was verified to significantly increase the solubilities of other water-insoluble natural polyphenols in aqueous solution. We suggest that addition of both HP and polar aprotic solvent in the reaction mixture is a powerful alternative to enhance production of polyphenolic chemicals rather than screening appropriate organic solvents for whole-cell catalysis of polyphenols.
Asunto(s)
Equol/biosíntesis , Microbiología Industrial/métodos , Solventes/química , Escherichia coli/genética , Isoflavonas/química , Agua/químicaRESUMEN
In this study, the optimum ammonium removal by activation of synthetic zeolite in the aqueous phase was investigated by batch ion exchange adsorption assay, and its surface changes due to activation modification was elucidated accordingly. Among the adsorbents examined, modified synthetic zeolite A-4 was the most effective at ammonium removal. The best activation condition of zeolite A-4 was established by Na+ and 300 °C heat treatment at pH around 6 to 7. Besides, the removal efficiency was investigated under various reaction conditions of pH, adsorbent dosage, stirring speed, and initial ammonium concentration. Finally, the adsorptive capacity Qe of synthetic zeolite A-4 activated by Na+ and heat treatment was determined as 31.9 mg/g at 1,000 mg-N/L of ammonium, whereas that of natural zeolite was measured as 16.0 mg/g. The obtained adsorption data was fitted to both Langmuir and Freundlich isotherm models, and the Langmuir isotherm model provided a better correspondence than the Freundlich isotherm. Finally, regeneration cycles for synthetic zeolite A-4 was determined for further industrial applications and efficient ammonium removal.
Asunto(s)
Compuestos de Amonio , Purificación del Agua , Zeolitas , Adsorción , Calor , Concentración de Iones de Hidrógeno , Intercambio Iónico , Cinética , Contaminantes Químicos del AguaRESUMEN
At least half of all stroke survivors experience fatigue; thus, it is a common cause of concern for patients, caregivers, and clinicians after stroke. This scientific statement provides an international perspective on the emerging evidence surrounding the incidence, prevalence, quality of life, and complex pathogenesis of poststroke fatigue. Evidence for pharmacological and nonpharmacological interventions for management are reviewed, as well as the effects of poststroke fatigue on both stroke survivors and caregivers.