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Cancer remains the leading cause of global mortality, prompting a paradigm shift in its treatment and outcomes with the advent of targeted therapies. Among the most prevalent mutations in RAS-driven cancers, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for approximately 86% of cases worldwide, particularly in lung, pancreatic, and colon cancers, contributing to poor prognosis and reduced overall survival. Despite numerous efforts to understand the biology of KRAS mutants and their pivotal role in cancer development, the lack of well-defined drug-binding pockets has deemed KRAS an "undruggable" therapeutic target, presenting significant challenges for researchers and clinicians alike. Through significant biochemical and technological advances, the last decade has witnessed promising breakthroughs in targeted therapies for KRAS-mutated lung, colon, and pancreatic cancers, marking a critical turning point in the field. In this chapter, we provide an overview of the characteristics of KRAS mutations across various solid tumors, highlighting ongoing cutting-edge research on the immune microenvironment, the development of KRAS-driven mice models, and the recent progress in the exploration of specific KRAS mutant-targeted therapeutic approaches. By comprehensive understanding of the intricacies of KRAS signaling in solid tumors and the latest therapeutic developments, this chapter will shed light on the potential for novel therapeutic strategies to combat KRAS-driven tumors and improve patient outcomes.
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Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Transducción de Señal , Humanos , Animales , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Transducción de Señal/efectos de los fármacos , Mutación , Terapia Molecular Dirigida , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Microambiente Tumoral/efectos de los fármacosRESUMEN
Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul-1 h-1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol-1, 100.59 kJ mol-1, 102.64 kJ mol-1 and 5.95 J mol-1 K-1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2-4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2-4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.
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Quitina , Quitinasas , Estabilidad de Enzimas , Oligosacáridos , Temperatura , Quitinasas/química , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Concentración de Iones de Hidrógeno , Quitina/química , Oligosacáridos/química , Quitosano/química , Especificidad por Sustrato , CinéticaRESUMEN
Supercapacitors are the latest development in the field of energy storage devices (ESDs). A lot of research has been done in the last few decades to increase the performance of supercapacitors. The electrodes of supercapacitors are modified by composite materials based on conducting polymers, metal oxide nanoparticles, metal-organic frameworks, covalent organic frameworks, MXenes, chalcogenides, carbon nanotubes (CNTs), etc. In comparison to rechargeable batteries, supercapacitors have advantages such as quick charging and high power density. This review is focused on the progress in the development of electrode materials for supercapacitors using composite materials based on conducting polymers, graphene, metal oxide nanoparticles/nanofibres, and CNTs. Moreover, we investigated different types of ESDs as well as their electrochemical energy storage mechanisms and kinetic aspects. We have also discussed the classification of different types of SCs; advantages and drawbacks of SCs and other ESDs; and the use of nanofibres, carbon, CNTs, graphene, metal oxide-nanofibres, and conducting polymers as electrode materials for SCs. Furthermore, modifications in the development of different types of SCs such as pseudo-capacitors, hybrid capacitors, and electrical double-layer capacitors are discussed in detail; both electrolyte-based and electrolyte-free supercapacitors are taken into consideration. This review will help in designing and fabricating high-performance supercapacitors with high energy density and power output, which will act as an alternative to Li-ion batteries in the future.
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Under tropical climate heat stress is a major challenge for livestock production. HSP70.1 is a ubiquitously expressed protein maintaining cellular machinery through proper folding of denatured proteins and prevents cellular apoptosis and protect cell from heat stress. Therefore, present investigation was undertaken to explore genetic variability in HSP70.1 gene in Gangatiri cattle, its comparison with buffalo sequences and differential expression in different season. The allelic variant was identified by sequencing amplified PCR product of HSP70.1 gene by primer walking. Season-wise total RNA samples was prepared for differential expression study. Brilliant SYBR Green QPCR technique was used to study the expression kinetics of this gene. DNA sequencing by primer walking identified four allelic variants in Gangatiri cattle. Sequence alignment study revealed four, six and one substitutions in the 5' untranslated region (5'UTR), coding and 3' untranslated region ((3'UTR) of HSP70.1 gene, respectively. Comparative analysis of HSP70.1 gene revealed that Cattle has shorter 5'UTR and 3' UTR than the buffalo. In Gangatiri cattle, summer season has significantly higher (P ≤ 0.05) expression of HSP70.1 than the spring and winter. The relative expression of HSP70.1 was increased by more than six folds in summer and nearly 1.5 folds higher in winter in comparison to the spring season. Therefore, HSP70.1 may be considered to have a critical role in the development of thermal tolerance in Gangatiri cattle.
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Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.
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Hematopoyesis , Hierro , Hematopoyesis/genética , Hierro/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Multipotentes/metabolismo , Regulación de la Expresión Génica , Diferenciación CelularRESUMEN
Bacterial cellulose (BC) is an exopolysaccharide produced by bacteria that has unusual structural features and is more refined than plant cellulose. BC has recently gained more attention in a variety of fields including biological and biomedical applications due to its excellent physiochemical properties including easy biodegradability, better water holding capacity, high tensile strength, high thermal stability, and high degree of polymerization. However, application of BC at industrial scale is still limited due to its high production cost and lesser yielding strains. The present study is an attempt to isolate and characterize a novel BC-producing bacterial strain. The bacterial strain S5 has resulted into maximum cellulose production of 4.76 ± 0.49 gL-1 (30°C, pH 7.0). The strain has been further identified as Stenotrophomonas sp. Derivation of nutritional and cultural conditions has resulted into 2.34-fold enhanced BC production (banana peel powder, peptone, tartaric acid, pH 7, 30°C). FTIR spectrum of BC revealed characteristic absorption bands which could be attributed to the O-H band, C-H stretching, C-O-C stretching band, O-H bending, and >CH2 bending, indicative of the ß-1,4 glycosidic linkages of cellulose. Thermogravimetric analysis has also revealed stability of polysaccharide backbones and characteristic weight loss points. Employment of banana peel powder has appeared as a proficient low-cost source for large-scale economic production of BC for industrial applications.
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Musa , Celulosa , Análisis Costo-Beneficio , Polvos , Bacterias/genéticaRESUMEN
OBJECTIVE: Pediatric irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with variable response to various therapeutic agents. Psyllium has been proven to be effective in adults; however, there is no study in children. The objective of this study is to evaluate the efficacy of psyllium husk as compared to placebo in pediatric IBS patients. METHODS: In this double-blind randomized controlled trial, 43 children were assigned to psyllium arm (Group A) and 38 into placebo arm (Group B). Severity is assessed at baseline and after 4 weeks of treatment using IBS severity scoring scale (IBS-SSS) and classified into mild, moderate, and severe categories. Categorical data was compared with chi-square test and paired categorical variable was compared with McNemer test. RESULTS: Mean ages (±SD; in years) of Groups A and B were 9.87 (2.7) and 9.82 (3.17), respectively, with median duration of illness of 12 months. At baseline, type, severity, and parameters (IBS-SSS) of IBS were equally distributed in 2 groups. There was a significant reduction in median interquartile range (IQR) of total IBS-SSS in psyllium versus placebo [75 (42.5-140) vs 225 (185-270); P < 0.001] at 4 weeks. Similarly 43.9% in Group A versus 9.7% in Group B attained remission [IBS-SSS < 75 ( P < 0.0001)]. The mean difference in IBS-SSS between Group A and Group B was -122.85 with risk ratio of 0.64 (95% CI; 0.42-0.83; P = 0.001) and absolute risk reduction of 32% (NNT = 3). CONCLUSIONS: Psyllium husk is effective for the therapy of pediatric IBS when compared with placebo in short term.
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Síndrome del Colon Irritable , Psyllium , Adulto , Humanos , Niño , Síndrome del Colon Irritable/complicaciones , Síndrome del Colon Irritable/tratamiento farmacológico , Psyllium/uso terapéutico , Método Doble Ciego , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Calidad de VidaRESUMEN
AgO, CoO, and ZnO (ACZ) mixed metal quantum dots (QDs) were synthesized by the sol-gel process. Polyaniline (PANI) was prepared by the chemical-oxidative technique. An in situ approach was used for the synthesis of ACZ decorated PANI plastic nanocomposites (NCs). TEM, FTIR, FESEM, UV-visible, DSC, Raman, photoluminescence, and XRD techniques were used for characterizing the QDs, PANI, and ACZ decorated PANI NCs. Experimental and theoretical (DFT) studies were used to support the results. NCs were studied for their adsorption, magnetic, photocatalytic, electrical, thermal, photoluminescence, antibacterial, and anticorrosive activities. The plastic NCs of size 35 nm (observed from XRD and TEM) were found to be paramagnetic. UV-visible spectroscopy and DFT techniques were used to observe the optical band gap of NCs and show an almost equal band gap i.e., 2.75 eV. In 1.0 M H2SO4, the NCs show an 82.0% corrosion inhibition efficiency for mild steel. The adsorption power of the silica gel + NCs packed column was higher than normal silica gel column. A very small low-intensity D band in the Raman spectra confirms defect-free NCs. The photocatalytic activity was observed against methyl-red dye in visible light. The thermal stability of plastic NCs was higher than pure PANI and QDs. The NCs were investigated for bactericidal activity against Gram (positive and negative) microorganisms. The ACZ decorated PANI NCs acted as good nanomaterials for adsorption, separation, magnetic, photocatalytic, photoluminescence, antibacterial, electrical, thermal insulator, and anticorrosive agent.
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MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/ß-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of ß-Catenin in a p53-independent manner. Wnt/ß-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/ß-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/ß-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.
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Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Proteómica/métodos , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiologíaAsunto(s)
Síndromes Mielodisplásicos , FN-kappa B , Humanos , Inflamación , Mielopoyesis , Transducción de SeñalRESUMEN
The present study was undertaken to investigate genetic variability in a fragment comprising 5'UTR along with partial coding sequence of Hsp70 gene and its association with thermotolerance traits in Murrah buffalo at ICAR-Research Complex for Eastern Region, Patna (India). The allelic variants were identified from genomic DNA samples using SSCP technique. The PCR products were sequenced and analyzed. Data on different thermotolerance traits recorded in three seasons were analyzed by least squares ANOVA taking the SSCP genotypes as fixed effect. Two allelic variants (A and B), each of 503-bp in size, were documented with frequency of 0.59 and 0.41, respectively, and three genotypes (AA, AB and BB) with corresponding frequency of 0.30, 0.58 and 0.12. The allelic variants were due to single nucleotide substitution at 55th base position leading to a change of threonine (A) to methionine (B) in amino acid sequence. Both the allelic variants had 99.8% similarity in nucleotide sequence. In phylogenetic tree, allele A was in a cluster while allele B and Gangatiri cattle sequence formed a different cluster. The SSCP genotypes had significant effect on different thermotolerance traits in summer with thermo-humidity index of ≥ 84. Buffaloes with AA genotype had the highest (P Ë 0.05) summer evening rectal temperature, respiration rate and pulse rate, inferring that the buffaloes carrying AA genotype had more stress in summer than those with AB and BB genotype. These SSCP genotypes might have differential role in heat shock protein response to induce thermotolerance of Murrah buffaloes in Gangetic plains.
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Regiones no Traducidas 5'/genética , Búfalos/genética , Proteínas HSP70 de Choque Térmico/genética , Termotolerancia/genética , Alelos , Animales , Variación Genética , Genotipo , India , Clima TropicalRESUMEN
This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/µl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.
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Búfalos , Proteínas HSP70 de Choque Térmico , Animales , Búfalos/genética , Búfalos/metabolismo , Expresión Génica , Perfilación de la Expresión Génica/métodos , Proteínas HSP70 de Choque Térmico/clasificación , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Calor , India , Estaciones del Año , Clima TropicalRESUMEN
Caspase-10 belongs to the class of initiator caspases and is a close homolog of caspase-8. However, the lack of caspase-10 in mice and limited substrate repertoire restricts the understanding of its physiological functions. Here, we report that ATP-citrate lyase (ACLY) is a caspase-10 substrate. Caspase-10 cleaves ACLY at the conserved Asp1026 site under conditions of altered metabolic homeostasis. Cleavage of ACLY abrogates its enzymatic activity and suppresses the generation of acetyl-CoA, which is critical for lipogenesis and histone acetylation. Thus, caspase-10-mediated ACLY cleavage results in reduced intracellular lipid levels and represses GCN5-mediated histone H3 and H4 acetylation. Furthermore, decline in GCN5 activity alters the epigenetic profile, resulting in downregulation of proliferative and metastatic genes. Thus caspase-10 suppresses ACLY-promoted malignant phenotype. These findings expand the substrate repertoire of caspase-10 and highlight its pivotal role in inhibiting tumorigenesis through metabolic and epigenetic mechanisms.
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ATP Citrato (pro-S)-Liasa/antagonistas & inhibidores , Carcinogénesis/patología , Caspasa 10/metabolismo , Epigénesis Genética/genética , Neoplasias/patología , Células A549 , Acetilcoenzima A/biosíntesis , Acetilación , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Lipogénesis/fisiología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante Heterólogo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Early and accurate diagnosis of pregnancy in animals is important for improving the reproductive management of livestock. The buffalo (Bubalus bubalis) is the most important dairy animal in India, but there are reproductive problems resulting from extended calving interval and ovulation occurring in the absence of behavioral estrus. The lack of simple methods for early pregnancy diagnosis intensifies these problems. The present study, therefore, was conducted to ascertain the role of the interferon-stimulated gene, (ISG), 15 in pregnancy detection. The anti-ISG15 Mab based ELISA was developed that could be used for detecting pregnancy at 18 to 20 days after artificial insemination (AI). The ISG15 protein was isolated from a pregnant buffalo and was amplified, and cloned in Escherichia coli by using coding region primers. The ISG15 gene was expressed in the host Escherichia coli BL21 (DE3), and the protocol was standardized for optimum gene expression. Using immortal hybridoma (fused myeloma and B cells) cells, a highly specific and sensitive antibody, anti-ISG15 mAb, for detecting ISG15 (protein) in the serum of pregnant buffaloes was obtained. A blocking ELISA was developed using the anti-ISG15 mAb to detect pregnancy in buffalo within 18 to 21 days after AI. The ISG15 gene was upregulated (P < 0.05) in pregnant buffalo at 18 to 21 days of pregnancy. This assay has an overall diagnostic accuracy of 75.0%. It, therefore, is concluded that recombinant ISG15 retains the potential for detecting pregnancy in B. bubalis and may have applications in ELISA kits for pregnancy detection in closely related species.
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Búfalos , Preñez/fisiología , Animales , Estro , Femenino , India , Inseminación Artificial , Interferones , Embarazo , Transcriptoma/fisiologíaRESUMEN
p53, the revered savior of genomic integrity, receives signals from diverse stress sensors and strategizes to maintain cellular homeostasis. However, the predominance of p53 overshadows the fact that this herculean task is no one-man show; rather, there is a huge army of regulators that reign over p53 at various levels to avoid an unnecessary surge in its levels and sculpt it dynamically to favor one cellular outcome over another. This governance starts right at the time of p53 translation, which is gated by proteins that bind to p53 mRNA and keep a stringent check on p53 protein levels. The same effect is also achieved by ubiquitylases and deubiquitylases that fine-tune p53 turnover and miRNAs that modulate p53 levels, adding precision to this entire scheme. In addition, extensive covalent modifications and differential protein interactions allow p53 to trigger a tailor-made response for a given circumstance. To magnify the marvel, these various tiers of regulation operate simultaneously and in various combinations. In this review, we have tried to provide a glimpse into this bewildering labyrinth. We believe that further studies will result in a better understanding of p53 regulation and that new insights will help unravel many aspects of cancer biology.
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Despite being one of the most well-studied transcription factors, the temporal regulation of p53-mediated transcription is not very well understood. Recent data suggest that target specificity of p53-mediated transactivation is achieved by posttranslational modifications of p53. K120 acetylation is a modification critical for recruitment of p53 to proapoptotic targets. Our data reveal that histone deacetylase 5 (HDAC5) binds to p53 and abrogates K120 acetylation, resulting in preferential recruitment of p53 to proarrest and antioxidant targets at early phases of stress. However, upon prolonged genotoxic stress, HDAC5 undergoes nuclear export. Concomitantly, p53 is acetylated at the K120 residue and selectively transactivates proapoptotic target genes, leading to onset of apoptosis. Furthermore, upon genotoxic stress in mice where HDAC5 expression is downregulated, the onset of apoptosis is accelerated in the highly vulnerable tissues. These findings suggest that HDAC5 is a key determinant of p53-mediated cell fate decisions in response to genotoxic stress.
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Acetilación/efectos de los fármacos , Apoptosis/genética , Daño del ADN/genética , Histona Desacetilasas/genética , Proteína p53 Supresora de Tumor/metabolismo , Transporte Activo de Núcleo Celular/genética , Adenoviridae/metabolismo , Adenoviridae/patogenicidad , Animales , Apoptosis/efectos de los fármacos , Etopósido/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HCT116 , Histona Desacetilasas/metabolismo , Humanos , Lisina/metabolismo , Ratones , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The paper deals with the exploitation of Ipomoea carnea as a feedstock for the production of bioethanol. Dilute acid pretreatment under optimum conditions (3%H2SO4, 120 °C for 45 min) produced 17.68 g L(-1) sugars along with 1.02 g L(-1) phenolics and 1.13 g L(-1) furans. A combination of overliming and activated charcoal adsorption facilitated the removal of 91.9% furans and 94.7% phenolics from acid hydrolysate. The pretreated biomass was further treated with a mixture of sodium sulphite and sodium chlorite and, a maximum lignin removal of 81.6% was achieved. The enzymatic saccharification of delignified biomass resulted in 79.4% saccharification with a corresponding sugar yield of 753.21 mg g(-1). Equal volume of enzymatic hydrolysate and acid hydrolysate were mixed and used for fermentation with a hybrid yeast strain RPRT90. Fermentation of mixed detoxified hydrolysate at 30 °C for 28 h produced ethanol with a yield of 0.461 g g(-1). A comparable ethanol yield (0.414 g g(-1)) was achieved using a mixture of enzymatic hydrolysate and undetoxified acid hydrolysate. Thus, I. carnea biomass has been demonstrated to be a potential feedstock for bioethanol production, and the use of hybrid yeast may pave the way to produce bioethanol from this biomass.
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Biocombustibles , Etanol/metabolismo , Ipomoea/metabolismo , Saccharomyces cerevisiae/genética , Ácidos , Biomasa , Quimera , Fermentación , Glucosa/metabolismo , Hidrólisis , Saccharomyces cerevisiae/metabolismo , Xilosa/metabolismoRESUMEN
The present work deals with the improvement of multiple stress tolerance in a glucose-xylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L(-1) furfural and 8 g L(-1) acetic acid. The mutant also showed good growth at high temperature (39-40°C). A study on the combined effect of multiple stresses during fermentation of glucose-xylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39°C) and inhibitor stress (0.25 g L(-1) vanillin, 0.5 g L(-1) furfural and 4 g L(-1) acetic acid), the mutant produced ethanol with a yield of 0.379 g g(-1), while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g(-1). Further, under the synergistic effect of sugar (250 g L(-1)), thermal (39°C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L(-1) ethanol by utilizing 162.42 g L(-1) of glucose-xylose mixture giving an ethanol yield of 0.295 g g(-1) and productivity of 0.57 g L(-1) h(-1). Under same condition the fusant RPR39 produced a maximum of 30.0 g L(-1) ethanol giving a yield and productivity of 0.21 g g(-1) and 0.42 g L(-1) h(-1) respectively. The molecular characterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast.
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Reactores Biológicos , Etanol/metabolismo , Fermentación/genética , Mutagénesis/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/genética , Ácido Acético/farmacología , Etanol/farmacología , Etanol/provisión & distribución , Fermentación/efectos de los fármacos , Fermentación/efectos de la radiación , Furaldehído/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Calor , Metilnitronitrosoguanidina/farmacología , Mutagénesis/efectos de los fármacos , Mutagénesis/efectos de la radiación , Mutágenos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/efectos de la radiación , Estrés Fisiológico/efectos de los fármacos , Rayos Ultravioleta , Xilosa/metabolismo , Xilosa/farmacologíaRESUMEN
The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose-xylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8 ± 0.31 g L(-1)), ethanol productivity (1.06 g L(-1) h(-1)) and ethanol yield (0.458 g g(-1)) by fermentation of glucose-xylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08 ± 0.142 g L(-1), ethanol yield of 0.44 g g(-1), productivity of 0.45 g L(-1) h(-1) and biomass yield of 0.40 g g(-1).