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A novel host-protein score (called MMBV) helps to distinguish bacterial from viral infection by combining the blood concentrations of three biomarkers: tumour necrosis factor related apoptosis inducing ligand (TRAIL), interferon gamma induced protein 10 (IP-10), and C-reactive protein (CRP). These host biomarkers are differentially expressed in response to bacterial versus viral acute infection. We conducted a prospective study, with a time series design, in healthy adult volunteers in the Netherlands. The aim was to determine the variability of TRAIL, IP-10, and CRP and the MMBV score in healthy adults across time. Up to six blood samples were taken from each healthy volunteer over a period of up to four weeks. In 77 healthy participants without recent or current symptoms, MMBV scores (maximal) were bacterial in 1.3 % and viral (or other non-infectious etiology) in 93.5 % of participants. There was little variation in the mean concentrations of TRAIL (74.5 pg/ml), IP-10 (113.6 pg/ml), and CRP (1.90 mg/L) as well as the MMBV score. The variability of biomarker measurement was comparable to the precision of the measurement platform for TRAIL, IP-10, and CRP. Our findings establish the mean values of these biomarkers and MMBV in healthy individuals and indicate little variability between and within individuals over time, supporting the potential utility of this novel diagnostic to detect infection-induced changes.
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Proteína C-Reactiva , Virosis , Adulto , Humanos , Proteína C-Reactiva/análisis , Quimiocina CXCL10 , Estudios Prospectivos , Ligandos , Biomarcadores , Factor de Necrosis Tumoral alfaRESUMEN
COVID-19 patients are oftentimes over- or under-treated due to a deficit in predictive management tools. This study reports derivation of an algorithm that integrates the host levels of TRAIL, IP-10, and CRP into a single numeric score that is an early indicator of severe outcome for COVID-19 patients and can identify patients at-risk to deteriorate. 394 COVID-19 patients were eligible; 29% meeting a severe outcome (intensive care unit admission/non-invasive or invasive ventilation/death). The score's area under the receiver operating characteristic curve (AUC) was 0.86, superior to IL-6 (AUC 0.77; p = 0.033) and CRP (AUC 0.78; p < 0.001). Likelihood of severe outcome increased significantly (p < 0.001) with higher scores. The score differentiated severe patients who further deteriorated from those who improved (p = 0.004) and projected 14-day survival probabilities (p < 0.001). The score accurately predicted COVID-19 patients at-risk for severe outcome, and therefore has potential to facilitate timely care escalation and de-escalation and appropriate resource allocation.
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COVID-19 , Humanos , Quimiocina CXCL10 , Unidades de Cuidados Intensivos , Curva ROC , Estudios Retrospectivos , PronósticoRESUMEN
Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as chiral building blocks for bioactive compounds and drugs. In this review article, we discuss the state of the art and development potential of epoxide hydrolases as biocatalysts based on the most recent approaches and techniques. The review covers new approaches to discover epoxide hydrolases using genome mining and enzyme metagenomics, as well as improving enzyme activity, enantioselectivity, enantioconvergence, and thermostability by directed evolution and a rational design. Further improvements in operational and storage stabilization, reusability, pH stabilization, and thermal stabilization by immobilization techniques are discussed in this study. New possibilities for expanding the synthetic capabilities of epoxide hydrolases by their involvement in non-natural enzyme cascade reactions are described.
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Epóxido Hidrolasas , Compuestos Epoxi , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/química , Catálisis , Compuestos Epoxi/química , Hidrólisis , Técnicas Genéticas , EstereoisomerismoRESUMEN
Maltooligosaccharides (MOS) are homooligosaccharides that consist of 3-10 glucose molecules linked by α-1,4 glycosidic bonds. As they have physiological functions, they are commonly used as ingredients in nutritional products and functional foods. Many researchers have investigated the potential applications of MOS and their derivatives in the pharmaceutical industry. In this review, we summarized the properties and methods of fabricating MOS and their derivatives, including sulfated and non-sulfated alkylMOS. For preparing MOS, different enzymatic strategies have been proposed by various researchers, using α-amylases, maltooligosaccharide-forming amylases, or glycosyltransferases as effective biocatalysts. Many researchers have focused on using immobilized biocatalysts and downstream processes for MOS production. This review also provides an overview of the current challenges and future trends of MOS production.
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Amilasas , Oligosacáridos , Oligosacáridos/química , alfa-Amilasas/metabolismo , Glucosa , BiocatálisisRESUMEN
OBJECTIVES: To determine the clinical utility and adverse consequences of routine admission chest x-ray (CXR) findings in patients with and without respiratory complaints and/or an abnormal chest examination. METHODS: In this prospective cohort study in an internal medicine department, we selected 273 patients and determined outcomes by chart review and physician interviews. The patients were divided into those with and without respiratory tract symptoms and/or findings on chest examination. The outcome variables were appropriate or inappropriate changes in treatment based on CXR findings. RESULTS: Of the 35 patients with respiratory tract symptoms/signs, 7 (20%) had a change in therapy based on CXR findings, which was effective in 5 of them. In the other 238 patients, an unexpected pleural empyema was detected in a hypotensive dialysis patient (0.4%, 95% confidence interval 0-2.3). Besides costs and radiation exposure, major adverse effects included two patients (0.8%, 95% confidence interval 0.1-3.0) with a false-positive test result that resulted in inappropriate hospitalizations and antibiotic therapy. In patients without respiratory tract symptoms or findings on physical examination, the clinical benefits and major adverse consequences were uncommon. CONCLUSIONS: Admission CXRs in patients without respiratory tract symptoms or findings are unwarranted except perhaps in older adult patients with comorbidities and an unclear admitting diagnosis.
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Admisión del Paciente/estadística & datos numéricos , Radiografía Torácica/estadística & datos numéricos , Enfermedades Respiratorias/diagnóstico por imagen , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Procedimientos Innecesarios/estadística & datos numéricosRESUMEN
Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aß)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.
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Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Nanoestructuras/química , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/uso terapéutico , Silicio/química , Animales , Supervivencia Celular/efectos de los fármacos , Ratones , Microscopía Confocal , Microscopía Fluorescente , Nanoestructuras/uso terapéutico , Factor de Crecimiento Nervioso/farmacocinética , Células PC12 , Porosidad , Ratas , Microtomografía por Rayos XRESUMEN
The clinical utility and adverse consequences of the admission and follow-up complete blood count (CBC) in hospitalised patients are unclear. We selected 273 patients chosen from a single internal medicine department. To determine clinical utility and adverse consequences, we interviewed attending physicians and reviewed patients' charts. There were 12 (4.4%) patients hospitalised because of the CBC test result, six referred appropriately with a low haemoglobin concentration found in outpatient clinics and six (2.2%) patients (95% confidence interval 0.8-4.7%) inappropriately hospitalised because of incidental findings. In the hospital, according to the physicians, nearly all treatment changes made were for blood transfusions that were not indicated in 18 (6.6%) patients (95% confidence interval 4.0-10.2%). The only unexpected findings were in four patients with an indication for a blood transfusion admitted with an acute coronary syndrome and haemoglobin values 8-9.9 g/dL, and in one bedridden patient with dementia with acute myeloid leukaemia. There were 290 follow-up CBC tests not resulting in differential treatment. We conclude that admission CBC tests commonly lead to adverse consequences, due to physician errors in judgement. Incidental findings of anaemia justify CBC testing in patients with an acute coronary event. The rare patient with an incidental finding resulting in appropriate differential treatment might justify non-selective admission CBC counts, if physician education reduces the rate of inappropriate blood transfusions.
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Hemoglobinas/análisis , Hospitalización/tendencias , Medicina Interna/tendencias , Uso Excesivo de los Servicios de Salud/tendencias , Anciano , Anciano de 80 o más Años , Anemia/sangre , Anemia/diagnóstico , Anemia/terapia , Recuento de Células Sanguíneas/normas , Recuento de Células Sanguíneas/tendencias , Transfusión Sanguínea/tendencias , Femenino , Estudios de Seguimiento , Humanos , Medicina Interna/normas , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production. RESULTS: Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 MutS strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5 l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated. CONCLUSIONS: There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes.
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Aspergillus niger/enzimología , Clonación Molecular , Monoaminooxidasa/aislamiento & purificación , Monoaminooxidasa/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Aspergillus niger/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Monoaminooxidasa/genética , Pichia/genética , Proteínas Recombinantes/genéticaRESUMEN
Biocatalyst immobilization is one of the techniques, which can improve whole cells or enzyme applications. This method, based on the fixation of the biocatalyst into or onto various materials, may increase robustness of the biocatalyst, allows its reuse, or improves the product yield. In recent decades, a number of immobilization techniques have been developed. They can be divided according to the used natural or synthetic material and principle of biocatalyst fixation in the particle. One option, based on the entrapment of cells or enzymes into a synthetic polyvinyl alcohol lens with original shape, is LentiKats® immobilization. This review describes the preparation principle of these particles and summarizes existing successful LentiKats® immobilizations. In addition, examples are compared with other immobilization techniques or free biocatalysts, pointing to the advantages and disadvantages of LentiKats®.
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Biotecnología/métodos , Células Inmovilizadas/metabolismo , Enzimas Inmovilizadas/metabolismo , Adhesión Celular , Alcohol Polivinílico/metabolismo , Unión ProteicaRESUMEN
This work demonstrates the first example of the immobilisation of MAO-N whole cells to produce a biocatalyst that remained suitable for repetitive use after 11 months of storage and stable up to 15 months after immobilisation. The production of Escherichia coli expressing recombinant MAO-N was scaled up to bioreactors under regulated, previously optimised conditions (10% DO, pH 7), and the amount of biomass was almost doubled compared to flask cultivation. Subsequently, pilot immobilisation of the whole-cell biocatalyst using LentiKats technology was performed. The amount of the immobilised biomass was optimised and the process was scaled up to a production level by immobilising 15 g of dry cell weight per litre of polyvinyl alcohol to produce 3 kg of whole-cell ready-to-use biocatalyst. The immobilised biocatalyst retained its initial activity over six consecutive biotransformations of the secondary amine model compound 3-azabicylo [3,3,0]octane, a building block of the hepatitis C drug telaprevir. Consecutive cultivation cycles in growth conditions not only increased the initial specific activity of biocatalyst produced on the industrial plant by more than 30%, but also significantly increased the rate of the biotransformation compared to the non-propagated biocatalyst.
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Células Inmovilizadas/metabolismo , Monoaminooxidasa/metabolismo , Monoaminas Biogénicas/metabolismo , Reactores Biológicos/microbiología , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Monoaminooxidasa/genética , Oxidación-Reducción , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Immobilization is one of the great tools for developing economically and ecologically available biocatalysts and can be applied for both enzymes and whole cells. Much research dealing with the immobilization of Escherichia coli has been published in the past two decades. E. coli in the form of immobilized biocatalyst catalyzes many interesting reactions and has been used mainly in laboratories, but also on an industrial scale, leading to the production of valuable substances. It has the potential to be applied in many fields of modern biotechnology. This paper aims to give a general overview of immobilization techniques and matrices suitable mostly for entrapment, encapsulation, and adsorption, which have been most frequently used for the immobilization of E. coli. An extensive analysis reviewing the history and current state of immobilized E. coli catalyzing different types of biotransformations is provided. The review is organized according to the enzymes expressed in immobilized E. coli, which were grouped into main enzyme classes. The industrial applications of immobilized E. coli biocatalyst are also discussed.
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Escherichia coli/metabolismo , Microbiología Industrial , Biocatálisis , Células Inmovilizadas/química , Células Inmovilizadas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Expresión GénicaRESUMEN
We aimed to investigate the cost-effectiveness of open surgery, compared to minimally invasive radical hysterectomy for early-stage cervical cancer, using updated survival data. Costs and utilities of each surgical approach were compared using a Markovian decision analysis model. Survival data stratified by surgical approach and surgery costs were received from recently published data. Average costs were discounted at 3%. The value of health benefits for each strategy was calculated using quality-adjusted life years (QALYs). Incremental cost-effectiveness ratio, calculated using the formula (average cost minimal invasive surgery-average cost open surgery)/(average QALY minimal invasive surgery-average QALY open surgery), was used for cost-effectiveness analysis. One-way sensitivity analysis was conducted for all variables. Open radical hysterectomy was found to be cost-saving compared to minimally invasive surgery with an incremental cost-effectiveness ratio of USD -66 and USD -373 for laparoscopic and robotic surgery, respectively. The most influential parameters in the model were surgery costs, followed by the disutility involved with open surgery. Until further data are generated regarding the survival of patients with early-stage cervical cancer treated by minimally invasive surgery, at current pricing, open radical hysterectomy is cost-saving compared to minimally invasive radical hysterectomy, both laparoscopic and robotic.
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The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.
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BACKGROUND: Treatment of severely ill COVID-19 patients requires simultaneous management of oxygenation and inflammation without compromising viral clearance. While multiple tools are available to aid oxygenation, data supporting immune biomarkers for monitoring the host-pathogen interaction across disease stages and for titrating immunomodulatory therapy is lacking. METHODS: In this single-center cohort study, we used an immunoassay platform that enables rapid and quantitative measurement of interferon γ-induced protein 10 (IP-10), a host protein involved in lung injury from virus-induced hyperinflammation. A dynamic clinical decision support protocol was followed to manage patients infected with severe acute respiratory syndrome coronavirus 2 and examine the potential utility of timely and serial measurements of IP-10 as tool in regulating inflammation. RESULTS: Overall, 502 IP-10 measurements were performed on 52 patients between 7 April and 10 May 2020, with 12 patients admitted to the intensive care unit. IP-10 levels correlated with COVID-19 severity scores and admission to the intensive care unit. Among patients in the intensive care unit, the number of days with IP-10 levels exceeding 1,000 pg/mL was associated with mortality. Administration of corticosteroid immunomodulatory therapy decreased IP-10 levels significantly. Only two patients presented with subsequent IP-10 flare-ups exceeding 1,000 pg/mL and died of COVID-19-related complications. CONCLUSIONS: Serial and readily available IP-10 measurements potentially represent an actionable aid in managing inflammation in COVID-19 patients and therapeutic decision-making. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04389645, retrospectively registered on May 15, 2020.
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COVID-19/sangre , Quimiocina CXCL10/sangre , Sistemas de Apoyo a Decisiones Clínicas , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , COVID-19/patología , COVID-19/terapia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Guías de Práctica Clínica como AsuntoRESUMEN
Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.
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Factor de Crecimiento Nervioso/administración & dosificación , Neuritas/fisiología , Neurogénesis/efectos de los fármacos , Neuronas/fisiología , Silicio/química , Animales , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , RatasRESUMEN
Bone morphogenetic proteins (BMPs) are probably the most important growth factors in bone formation and healing. However, the utilization of BMPs in clinical applications is mainly limited due to the protein poor solubility at physiological pH, rapid clearance and relatively short biological half-life. Herein, we develop degradable porous silicon (PSi)-based carriers for sustained delivery of BMP-2. Two different loading approaches are examined, physical adsorption and covalent conjugation, and their effect on the protein loading and release rate is thoroughly studied. The entrapment of the protein within the PSi nanostructures preserved its bioactivity for inducing osteogenic differentiation of rabbit bone marrow mesenchymal stems cells (BM-MSCs). BM-MSCs cultured with the BMP-2 loaded PSi carriers exhibit a relatively high alkaline phosphatase (ALP) activity. We also demonstrate that exposure of MSCs to empty PSi (no protein) carriers generates some extent of differentiation due to the ability of the carrier's degradation products to induce osteoblast differentiation. Finally, we demonstrate the integration of these promising BMP-2 carriers within a 3D-printed patient-specific implant, constructed of poly(caprolactone) (PCL), as a potential bone graft for critical size bone defects.
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Although nerve growth factor (NGF) is beneficial for the treatment of numerous neurological and non-neurological diseases, its therapeutic administration represents a significant challenge, due to the difficulty to locally deliver relevant doses in a safe and non-invasive manner. In this work, we employ degradable nanostructured porous silicon (PSi) films as carriers for NGF, allowing its continuous and prolonged release, while retaining its bioactivity. The PSi carriers exhibit high loading efficacy (up to 90%) of NGF and a continuous release, with no burst, over a period of>26days. The released NGF bioactivity is compared to that of free NGF in both PC12 cells and dissociated dorsal root ganglion (DRG) neurons. We show that the NGF has retained its bioactivity and induces neurite outgrowth and profound differentiation (of >50% for PC12 cells) throughout the period of release within a single administration. Thus, this proof-of-concept study demonstrates the immense therapeutic potential of these tunable carriers as long-term implants of NGF reservoirs and paves the way for new localized treatment strategies of neurodegenerative diseases.
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Preparaciones de Acción Retardada/química , Nanoestructuras/química , Factores de Crecimiento Nervioso/administración & dosificación , Neuronas/efectos de los fármacos , Silicio/química , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/farmacología , Neurogénesis/efectos de los fármacos , Neuronas/citología , Células PC12 , Porosidad , RatasRESUMEN
Pichia pastoris is currently one of the most preferred microorganisms for recombinant enzyme production due to its efficient expression system. The advantages include the production of high amounts of recombinant proteins containing the appropriate posttranslational modifications and easy cultivation conditions. α-L-Rhamnosidase is a biotechnologically important enzyme in food and pharmaceutical industry, used for example in debittering of citrus fruit juices, rhamnose pruning from naringin, or enhancement of wine aromas, creating a demand for the production of an active and stable enzyme. The production of recombinant α-L-rhamnosidase cloned in the Mut(S) strain of P. pastoris KM71H was optimized. The encoding gene is located under the control of the AOX promoter, which is induced by methanol whose concentration is instrumental for these strain types. Fermentation was upscaled in bioreactors employing various media and several methanol-feeding strategies. It was found that fed batch with BSM media was more effective compared to BMMH (Buffered Methanol-complex Medium) media due to lower cost and improved biomass formation. In BSM (Basal Salt Medium) medium, the dry cell weight reached approximately 60 g/L, while in BMMH it was only 8.3 g/L, without additional glycerol, which positively influenced the amount of enzyme produced. New methanol feeding strategy, based on the level of dissolved oxygen was developed in this study. This protocol that is entirely independent on methanol monitoring was up scaled to a 19.5-L fermenter with 10-L working volume with the productivity of 13.34 mgprot/L/h and specific activity of α-L-rhamnosidase of 82 U/mg. The simplified fermentation protocol was developed for easy and effective fermentation of P. pastoris Mut(S) based on dissolved oxygen monitoring in the induction phase of an enzyme production.
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The bacterial strains of the genus Nocardia were used for the bioconversion of fumaric acid to L-malic acid. The ability of the bacterial strain Nocardia sp. CCM 4837/A to produce L-malic acid from fumaric acid was investigated under various conditions. The optimal temperature for the bioconversion was approximately 37 °C, and the optimal pH was around 8.0. The addition of an inductor (fumarate salt) to the fermentation medium was necessary to enhance enzyme activity. The presence of detergent Triton X-100 (0.02-0.1 %) in the reaction mixture rapidly increased the enzyme activity of fumarase. The specific fumarase activity of intact cells Nocardia sp. CCM 4837/A increased from 2.8 to 75 U/mg after optimising the experimental conditions described here. Pretreatment of the Nocardia cells with malonate was not necessary because succinate was not detected as a by-product under our experimental conditions.
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Fumarato Hidratasa , Fumaratos/química , Malatos/química , Fumaratos/metabolismo , Malatos/metabolismo , Nocardia/química , Nocardia/enzimología , Nocardia/metabolismoRESUMEN
Clostridium acetobutylicum immobilised in polyvinylalcohol, lens-shaped hydrogel capsules (LentiKats(®)) was studied for production of butanol and other products of acetone-butanol-ethanol fermentation. After optimising the immobilisation protocol for anaerobic bacteria, continuous, repeated batch, and fed-batch fermentations in repeated batch mode were performed. Using glucose as a substrate, butanol productivity of 0.41 g/L/h and solvent productivity of 0.63 g/L/h were observed at a dilution rate of 0.05 h(-1) during continuous fermentation with a concentrated substrate (60 g/L). Through the process of repeated batch fermentation, the duration of fermentation was reduced from 27.8h (free-cell fermentation) to 3.3h (immobilised cells) with a solvent productivity of 0.77 g/L/h (butanol 0.57 g/L/h). The highest butanol and solvent productivities of 1.21 and 1.91 g/L/h were observed during fed-batch fermentation operated in repeated batch mode with yields of butanol (0.15 g/g) and solvents (0.24 g/g), respectively, produced per gram of glucose.