RESUMO
Accurate and reliable estimation of specific growth rate (µ) in real-time is pivotal for reliable process monitoring of a bioprocess and subsequent implementation of advanced control strategies. Gibbs free energy dissipation is imminent for any biological system, and the metabolic heat flow measurements (calorimetry) formed the basis for estimating µ. However, the rationale behind selecting a suitable µ estimator model based on calorimetric perspective remains unexplored. The present investigation addresses the notion behind the selection of an appropriate estimator for µ and the assessment of the estimator models was illustrated using different types of energy metabolism, namely, high exothermic and low exothermic processes. The results indicated that the µ values from the instantaneous heat flow estimator significantly deviated (10-fold higher) from the offline values for highly exothermic process. Notably, the cumulative heat-based estimator accurately estimated µ values on both types of energy metabolism with performance metrics <0.005 h-1.
Assuntos
Calorimetria , Metabolismo Energético , Modelos Biológicos , Temperatura Alta , TermodinâmicaRESUMO
Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin-an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30 g L-1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N-acetylglucosamine (S2) resulted in a significant 1.6-fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi-substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method.
RESUMO
During the epoch of sustainable development, leveraging cellular systems for production of diverse chemicals via fermentation has garnered attention. Industrial fermentation, extending beyond strain efficiency and optimal conditions, necessitates a profound understanding of microorganism growth characteristics. Specific growth rate (SGR) is designated as a key variable due to its influence on cellular physiology, product synthesis rates and end-product quality. Despite its significance, the lack of real-time measurements and robust control systems hampers SGR control strategy implementation. The narrative in this contribution delves into the challenges associated with the SGR control and presents perspectives on various control strategies, integration of soft-sensors for real-time measurement and control of SGR. The discussion highlights practical and simple SGR control schemes, suggesting their seamless integration into industrial fermenters. Recommendations provided aim to propose new algorithms accommodating mechanistic and data-driven modelling for enhanced progress in industrial fermentation in the context of sustainable bioprocessing.
Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Fermentação , Microbiologia Industrial , Reatores Biológicos/microbiologia , Microbiologia Industrial/métodos , Algoritmos , Bactérias/metabolismo , Bactérias/crescimento & desenvolvimentoRESUMO
Augmentation of the activity of Food and Drug Administration-approved antibiotics by an adjuvant or antibiotic carrier is considered one of the promising strategies to fight against antibiotic-resistant bacteria. This study reports the development of sulfonium-cross-linked hyaluronic acid (HA)-based polymer (HA-SS-HA) as an inherent antimicrobial agent and antibiotic carrier. The HA-SS-HA polymer offers the potential for encapsulating various classes of antibiotics and accomplishing a stimuli-responsive release profile in the presence of hyaluronidase produced by bacterial cells within their extracellular environment. Systematic antibacterial studies reveal that the HA-SS-HA-encapsulated antibiotics (vancomycin, amoxicillin, and tetracycline) restore its activity against the antibiotic-resistant bacterial cells methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), and Pseudomonas aeruginosa. The HA-SS-HA gel shows robust efficacy in eradicating the mature biofilm of Staphylococcus aureus (S. aureus). The membrane-disrupting activity reveals that HA-SS-HA can also counteract the antibiotic resistance mechanism of the bacterial cells. The in vivo studies reveal excellent wound-healing activity of HA-SS-HA in albino laboratory-bred (BALB/c) mice. The outcome of additional antibacterial studies reveals that antibiotics-encapsulated HA-SS-HA hydrogel can effectively combat Gram-negative, Gram-positive, and antibiotic-resistant bacterial strains. Therefore, revitalizing the activity of commercial antibiotics by HA-SS-HA can be considered a valuable and economically effective strategy to fight against antibiotic-resistant bacteria.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Estados Unidos , Animais , Camundongos , Antibacterianos/farmacologia , Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Staphylococcus aureus , Bactérias , Portadores de Fármacos/farmacologia , PolímerosRESUMO
Lack of appropriate process models, reliable online sensors, and process variability in bioprocess systems are poising challenges in real-time monitoring and control of critical process parameters (CPPs). This present investigation deals with the development of a non-invasive soft sensor by utilizing metabolic heat rate as input signal for online estimation of specific growth rate (µest) during the induction phase of glycoengineered Pichia pastoris for human interferon-alpha 2b (huIFNα2b) production. Feedforward strategy employing a predetermined exponential feeding of methanol during the induction phase was dealt at defined setpoint values (µSP). Standard PID controller with predetermined gain values regulated methanol feeding in accordance with the deviation from the µSP value. An adaptive PID (gain scheduling) significantly minimized the deviation of µ from its µSP value, reduced the amplitude of oscillation and achieved long-term controller stability. Robust control of methanol feeding by adaptive PID resulted in a 1.5 and 2.2-fold increase in productivity of huIFNα2b compared to standard PID and feedforward controls respectively. Moreover, adaptive PID control facilitated narrow range control of µ for longer durations (> 20 h) with a low average tracking error (< 6%) enumerating its scope of application in therapeutic protein production in near future.
Assuntos
Metanol , Pichia , Humanos , Pichia/metabolismo , Metanol/metabolismo , Fermentação , Temperatura Alta , Interferon alfa-2/metabolismo , Reatores Biológicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
D-Pantothenic acid (DPA), also known as vitamin B5 is associated with several biological functions and its deficiency causes metabolic and energetic disorders in humans. Fortification of foods with DPA is the viable option to address this risk. DPA biological production route employs pantoate-ß-alanine ligase (PBL) as the key enzyme, which avoids the tedious and time-consuming optical resolution process. The selection of an efficient PBL enzyme is vital for the biological production of DPA. In this study, the panC gene encoding PBL from Escherichia coli, Bacillus megaterium, Corynebacterium glutamicum and Bacillus subtilis was expressed in B. megaterium. B. subtilis derived panC exhibited high PBL activity 61.62 ± 2.15 U/mL. Co-expression of phosphoenolpyruvate carboxykinase (pckA) did not improve the DPA production in B. megaterium. Biocatalytic fed-batch fermentation with externally supplemented precursor substrates (D-pantoic acid and ß-alanine) improved DPA titer to 45.56 ± 0.53 g/L. Daily dietary requirements of DPA for different age groups (including babies, small children, athletes and elderly people) is steadily increasing and the improved DPA production addressed in this study offers advantage for its application in fortification of food products meeting the emerging nutritional demand. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05093-6.
RESUMO
High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study, Bacillus megaterium was metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08 g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, ß-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-ß-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05 g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of ß-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53 g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinant B. megaterium.
Assuntos
Bacillus megaterium , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Fermentação , Engenharia Metabólica/métodos , Ácido Pantotênico/genética , Ácido Pantotênico/metabolismo , beta-Alanina/genética , beta-Alanina/metabolismoRESUMO
3-Aminopropionic acid (3-APA) has wide applications in food, cosmetics, pharmaceuticals, chemical, and polymer industries. This present study aimed to develop an eco-friendly whole-cell biocatalytic process for the bio-production of 3-APA from fumaric acid (FA) using Bacillus megaterium. A dual-enzyme cascade route with aspartate-1-decarboxylases (ADC) from Bacillus subtilis and native aspartate ammonia-lyase (AspA) was developed. Divergent catalytic efficiencies between these two enzymes led to an imbalance between both enzyme reactions. In order to coordinate AspA and ADC expression levels, gene mining, optimization, and duplication strategies were employed. Additionally, culture cultivation conditions and biocatalysis process parameters were optimized. A maximum 3-APA titer was obtained (11.68 ± 0.26 g/L) with a yield of 0.78 g/g under the following optimal conditions: 45 °C, pH 6.0, and 15 g/L FA. This study established a biocatalysis process for the production of 3-APA from FA using the whole cells of the recombinant B. megaterium.
Assuntos
Aspartato Amônia-Liase , Bacillus megaterium , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Escherichia coli/genética , Fumaratos , beta-AlaninaRESUMO
This present investigation addressing the metabolic bottleneck in synthesis of high MW HA by Streptococcus zooepidemicus and illustrates the application of calorimetric fed-batch control of µ at a narrower range. Feedforward (FF) and feedback (FB) control was devised to improve the molecular weight (MW) of HA production by S. zooepidemicus. Metabolic heat measurements (Fermentation calorimetry) were modeled to decipher real-time specific growth rate, [Formula: see text] was looped into the PID circuit, envisaged to control [Formula: see text] to their desired setpoint values 0.05 [Formula: see text], 0.1 [Formula: see text], and 0.15 [Formula: see text] respectively. Similarly, a predetermined exponential feed rate irrespective of real-time µ was carried out in FF strategy. The developed FB strategy established a robust control capable of maintaining the specific growth rate (µ) close to the [Formula: see text] value with a minimal tracking error. Exponential feed rate carried out with a lowest [Formula: see text] of 0.05 [Formula: see text] showed an improved MW of HA to 2.98 MDa and 2.94 MDa for the FF and FB-based control strategies respectively. An optimal HA titer of 4.73 g/L was achieved in FF control strategy at [Formula: see text]. Superior control of µ at low [Formula: see text] value was observed to influence HA polymerization positively by yielding an improved MW and desired polydispersity index (PDI) of HA. PID control offers advantage over conventional fed-batch method to synthesize HA at an improved MW. Calorimetric signal-based µ control by PID negates adverse effects due to the secretion of other end products albeit maintaining regular metabolic activities. KEY POINTS: First report to compare HA productivities by feedforward and feedback control strategy. Inherent merits of regulating µ at narrower range were entailed. Relationship between operating µ and HA molecular weight was discussed.
Assuntos
Streptococcus equi , Fermentação , Temperatura Alta , Ácido Hialurônico , Peso MolecularRESUMO
Real-time estimation of physiological properties of the cell during recombinant protein production would ensure enhanced process monitoring. In this study, we explored the application of dielectric spectroscopy to track the fed-batch phase of recombinant Escherichia coli cultivation for estimating the physiological properties, namely, cell diameter and viable cell concentration (VCC). The scanning capacitance data from the dielectric spectroscopy were pre-processed using moving average. Later, it was modeled through a nonlinear theoretical Cole-Cole model and further solved using a global evolutionary genetic algorithm (GA). The parameters obtained from the GA were further applied for the estimation of the aforementioned physiological properties. The offline cell diameter and cell viability data were obtained from particle size analyzer and flow cytometry measurements to validate the Cole-Cole model. The offline VCC was calculated from the cell viability % from flow cytometry data and dry cell weight concentration. The Cole-Cole model predicted the cell diameter and VCC with an error of 1.03% and 7.72%, respectively. The proposed approach can enable the operator to take real-time process decisions to achieve desired productivity and product quality.
Assuntos
Espectroscopia Dielétrica , Escherichia coli , Sobrevivência Celular , Espectroscopia Dielétrica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Teóricos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In this study, we aimed to develop a Bacillus megaterium based whole-cell biocatalyst for the bio-production of 3-aminopropionic acid (3-APA). l-aspartate-α-decarboxylases (ADC) (EC: 4.1.1.11) from Escherichia coli, B. megaterium, Corynebacterium glutamicum, and Bacillus subtilis were expressed in B. megaterium. B. subtilis derived ADC (panD Bs ) exhibited the highest ADC activity of 0.9 ± 0.02 U/mL in recombinant B. megaterium. Combination of codon optimization and gene duplication strategies resulted in 415.56% enhancement of ADC activity compared to panD Bs . The culture growth conditions of B. megaterium (BMD-7) for 3-APA production were optimized as follows: inducer concentration, 0.5% (w/v); time of induction, 3 h; induction temperature, 37 °C and post-induction incubation time, 8 h. Improvement of the whole-cell biocatalytic process efficiency, was dealt by optimization of reaction temperature, reaction pH, metal ion additives and l-aspartic acid concentration. Shake flask level experiments yielded an enhanced 3-APA titer (16.18 ± 0.26 g/L) and a yield of 0.89 g/g under optimized conditions viz., 45 °C, pH 6.0 and 20 g/L of l-aspartic acid. This study demonstrates the potential of B. megaterium for 3-APA production and paves the scope for the development of 3-APA producing strains in near future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02885-7.
RESUMO
The devastating antibacterial infections, coupled with their antibiotic resistance abilities, emphasize the need for effective antibacterial therapeutics. In this prospect, liposomal delivery systems have been employed in improving the efficacy of the antibacterial agents. The liposome-based antibiotics enhance the therapeutic potential of the new or existing antibiotics and reduce their adverse effects. The current study describes the development of sulfonium-based antibacterial lipids that demonstrate the delivery of existing antibiotics. The presence of cationic sulfonium moieties and inherent membrane targeting abilities of the lipids could help reduce the antibiotic resistance abilities of the bacteria and deliver the antibiotics to remove the infectious pathogens electively. The transmission electron microscopic images and dynamic light scattering analyses revealed the liposome formation abilities of the sulfonium-based amphiphilic compounds in the aqueous medium. The effectiveness of the compounds was tested against the Gram-negative and Gram-positive bacterial strains. The viability of the bacterial cells was remarkably reduced in the presence of the compounds. The sulfonium-based compounds with pyridinium moiety and long hydrocarbon chains showed the most potent antibacterial activities among the tested compounds. Mechanistic studies revealed the membrane-targeted bactericidal activities of the compounds. The potent compound also showed tetracycline and amoxicillin encapsulation and sustained release profiles in the physiologically relevant medium. The tetracycline and amoxicillin-encapsulated lipid showed much higher antibacterial activities than the free antibiotics at similar concentrations, emphasizing the usefulness of the synergistic effect of sulfonium-based lipid and the antibiotics, signifying that the sulfonium lipid penetrated the bacterial membrane and increased the cellular uptake of the antibiotics. The potent lipid also showed therapeutic potential, as it is less toxic to mammalian cells (like HeLa and HaCaT cells) at concentrations higher than their minimum inhibitory concentration values against S. aureus, E. coli, and MRSA. Hence, the sulfonium-based lipid exemplifies a promising framework for assimilating various warheads, and provides a potent antibacterial material.
RESUMO
Chitosan was fabricated via gelation method using CaBr2.xH2O/methanol solution and was studied as a potential adsorbent (MCh) in adsorbing anionic synthetic dyes like Bromophenol blue (BB), Direct blue 6 (DB) and Congo red (CR) from single (one dye species at a time) and multi (having two dyes; binary and all three dyes; tertiary) adsorptive systems. Physico-chemical modifications of MCh surface prior and post modification and dye adsorption were evaluated using scanning electron microscopy, Energy-dispersive X-ray spectroscopy, powder X-ray diffraction analysis, surface area analysis and Fourier-transformed infrared spectroscopy. Influential parameters influencing the adsorption process viz. initial pH of dye solution, MCh dosage, adsorption temperature and initial concentration of dye species were optimised. Adsorptive studies involving single adsorptive setups verified formation of sorbate's (dye species) monolayer over the sorbent's (MCh) surface via chemisorption; as established by Langmuir isotherm and pseudo-second order kinetics model analysis. Theoretical maximum adsorption capacities of MCh for BB, DB and CR was found to be 81.301 mg/g, 163.934 mg/g and 75.758 mg/g, respectively. Meanwhile, for all multi-adsorptive systems, competitive Langmuir isotherm model verified antagonistic behaviour of an individual dye over other dye adsorption over MCh surface in their respective adsorptive systems. Thermodynamics of the sorbate-sorbent interaction was exothermic, spontaneous, with elevated degree of disorderedness; concluding the interaction as thermodynamically favourable. Co-existing metal cations and anionic salts had minimal effect on MCh's adsorption efficiency. Phytotoxicity assay via germination of Vigna mungo seeds verified the efficacy of the adsorbent in eliminating the dye species from single and multi-adsorptive systems.
Assuntos
Quitosana , Poluentes Químicos da Água , Adsorção , Corantes , Concentração de Íons de Hidrogênio , Cinética , Porosidade , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , Poluentes Químicos da Água/análiseRESUMO
Heparosan, a capsular polysaccharide synthesized by certain pathogenic bacteria, is a promising precursor for heparin production. Heparosan production is catalyzed by the formation of KfiC-KfiA complex and the subsequent action of KfiC and KfiA proteins. Polycistronic expression of kfiC and kfiA in Bacillus megaterium yielded an unbalanced expression of KfiC and KfiA proteins resulted in decreased heparosan production. In this study, dual promoter plasmid system was constructed to increase the expression levels of KfiC and KfiA proteins. Dual promoter plasmid system along with UDP-glucuronic acid pathway overexpression (CADuet-DB) increased the heparosan production to 203 mg/L in shake flask experiments. Batch fermentation of strain CADuet-DB under controlled conditions yielded a maximum heparosan concentration of 627 mg/L, which is 59% higher than strain CA-DB. A modified logistic model is applied to describe the kinetics of heparosan production and biomass growth. Fed batch fermentation resulted in 3-fold enhancement in heparosan concentration (1.96 g/L), compared to batch fermentation. Nuclear magnetic resonance analysis revealed that heparosan from strain CADuet-DB was similar to Escherichia coli K5 heparosan. These results suggested that dual promoter expression system is a promising alternative to polycistronic expression system to produce heparosan in B. megaterium.
Assuntos
Bacillus megaterium , Dissacarídeos , Proteínas de Escherichia coli , Escherichia coli/genética , Expressão Gênica , Glicosiltransferases , N-Acetilglucosaminiltransferases , Regiões Promotoras Genéticas , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Dissacarídeos/biossíntese , Dissacarídeos/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Glicosiltransferases/biossíntese , Glicosiltransferases/genética , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/genéticaRESUMO
Fermentative poly-3-hydroxybutyrate (PHB) production is mainly limited by the cost of raw material. In this present study, low-cost feedstock viz., millet bran residue (MBRH) and rapeseed meal hydrolysates were successfully utilized for PHB production. Metabolic engineering of Bacillus megaterium by co-expression of both precursor (phbRBC) and NADPH cofactor regeneration (zwf) genes resulted in 2.67-fold enhancement in PHB accumulation compared to wild strain. Modified logistic model characterized B.megaterium growth and PHB production effectively. The kinetic analysis proved that maximum cell concentration (15.01 g.L-1) and growth-associated constant (0.22 g.g-1) were found to be higher for initial MBRH concentration (S0 = 20 g.L-1). PHB production kinetics elucidated its expression in B.megaterium was growth-associated. PHB synthesized by B.megaterium was characterized using FTIR, NMR, XRD, DSC/TGA, FESEM and the physio-chemical properties enumerated its as a potential biodegradable plastic for industrial application.
Assuntos
Bacillus megaterium , Brassica napus , Ácido 3-Hidroxibutírico , Hidroxibutiratos , Cinética , Milhetes , PoliésteresRESUMO
Diclofenac (DCF), a persistent pharmaceutical micropollutant which occurs in the ecosystems causing adverse effects on aquatic as well as terrestrial organisms. In this study, magnetic sawdust (MSD) was prepared using co-precipitation method for biosorptive removal of DCF from water. The MSD was characterized using various analytical techniques like microscopic and spectroscopic analysis. Magnetometer study confirms the ferromagnetic behavior of the biosorbent which is a key advantage in the separation of MSD after biosorption. The effect of experimental parameters was optimized in batch mode with evaluated maximum efficiency of 86.12 % at pH 6, biosorbent dosage 25 mg for 50 mg/L of DCF. Ecotoxicological assessment has been performed for the treated and untreated sample using plant seeds, microbes and zebra fish to check the adverse effects of DCF on these organisms. Evaluation of toxicity studies revealed that inhibition concentration of DCF for various seeds (60.91 mg/L to 43.11 mg/L), E. coli (48.82 µg/mL) and B. subtilis (31.55 µg/mL). The lethal concentration of DCF on the Danio rerio was found to be 156.99 mg/L. In contrast, significant increase in both the concentration measures of DCF after biosorption was observed making this biosorbent a potent alternative to other available treatment measures.
Assuntos
Diclofenaco , Poluentes Químicos da Água , Animais , Diclofenaco/toxicidade , Ecossistema , Escherichia coli , Fenômenos Magnéticos , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Low molecular weight heparosan is an un-sulfated polysaccharide primarily used as a precursor for heparin synthesis that has recently been used in drug delivery applications. Heparosan synthesis from recombinant bacterial systems provides a safer alternative to naturally producing pathogenic bacterial systems. In this study, we engineered a functional heparosan synthesis pathway in Bacillus megaterium by the expression of E. coli K5 kfiC and kfiA glycosyltransferase genes. Upregulation of individual UDP-sugar precursor pathway genes enhanced the heparosan production, indicating that UDP-precursor sugar concentrations were limiting the biosynthesis. The engineered B. megaterium yielded a maximum heparosan concentration of 394 mg/L in batch bioreactor. The heparosan titer was further increased to 1.32 g/L in fed-batch fermentation. Nuclear magnetic resonance analysis revealed that the chemical structure of B. megaterium derived heparosan was identical to E. coli K5 heparosan. The heparosan molecular weight varied from 31 to 60 kDa, indicating its potential as a precursor for chemoenzymatic heparin biosynthesis. This study provides an efficient process to produce heparosan from non-pathogenic B. megaterium.
Assuntos
Bacillus megaterium/genética , Dissacarídeos/genética , Escherichia coli/genética , Glicosiltransferases/genética , Vias Biossintéticas/genética , Proteínas de Escherichia coli/genética , Fermentação/genética , Engenharia Metabólica/métodos , Peso Molecular , N-Acetilglucosaminiltransferases/genéticaRESUMO
D-lactic acid (DLA) serves as a key monomer enhancing both the mechanical and thermal properties of Poly(lactic) acid films and coatings, extensively used in the food packaging industry. Economically viable production of optically pure DLA by Lactobacillus delbrueckii NBRC3202 was achieved using a low-cost carbon source, Kodo millet bran residue hydrolysate (KMBRH) and nitrogen source (casein enzyme hydrolysate (CEH) resulting in a high DLA yield of 0.99 g g-1 and KMBRH conversion to final product (95.3%). The optimum values for kinetic parameters viz., specific growth rate (0.11 h-1), yield coefficient of biomass on KMBRH (0.10 g g-1) and DLA productivity (0.45 g L-1 h-1) were achieved at 5 g L-1 of CEH dosage under controlled pH environment. A comparative study and kinetic analysis of different neutralizing agents (NaOH, NH3, CaCO3 and NaHCO3) under pH controlled environment for KMBRH based DLA production was addressed effectively through bioreactor scale experiments. Maximum cell concentration (1.29 g L-1) and DLA titer (45.08 g L-1) were observed with NH3 as a neutralizing agent. Kinetic analysis of DLA production under different neutralization agents demonstrated that the logistic derived model predicted biomass growth, KMBRH consumption and DLA production efficiently (R 2 > 0.92).
RESUMO
The present study is focused on systematic process and kinetic investigation of hyaluronic acid (HA) production strategy unraveling the role of dissolved oxygen (DO) and N-acetyl glucosamine (GlcNAc) towards the enhancement of HA titer and its molecular weight. Maintaining excess DO levels (10-40% DO) through DO-stat control and the substitution of GlcNAc at a range (5-20 g/L) with glucose (Glc) critically influenced HA production. DO-stat control strategy yielded a promising HA titer (2.4 g/L) at 40% DO concentration. Controlling DO level at 20% (DO-stat) was observed to be optimum resulting in a significant HA production (2.1 g/L) and its molecular weight ranging 0.98-1.45 MDa with a consistent polydispersity index (PDI) (1.57-1.69). Substitution of GlcNAc with Glc at different proportions explicitly addressed the metabolic trade-off between HA titer and its molecular weight. GlcNAc substitution positively influenced the molecular weight of HA. The highest HA molecular weight (2.53 MDa) of two-fold increase compared with glucose as sole carbon substrate and narrower PDI (1.35 ± 0.18) was achieved for the 10:20 (Glc:GlcNAc) proportion. A novice attempt on modeling the uptake of dual substrates (Glc and GlcNAc) by Streptococcus zooepidemicus for HA production was successfully accomplished using double Andrew's growth model and the kinetic parameters were estimated reliably.
Assuntos
Acetilglucosamina/metabolismo , Ácido Hialurônico/biossíntese , Oxigênio/metabolismo , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/metabolismo , Biomassa , Fermentação , Glucose/metabolismo , Cinética , Peso MolecularRESUMO
Real-time monitoring of glycoengineered Pichia pastoris by employing process analytical technology (PAT) tools is vital for gaining deeper insights into the therapeutic protein production process. The present study focuses on influence of mixed feed carbon substrates during the induction phases of glycoengineered P. pastoris cultivation, for recombinant human interferon α2b (huIFNα2b) production by employing calorimetric (biological heat rate, q B ) and respirometric (oxygen uptake rate and carbon dioxide evolution rate) measurements. Mixed feed stream of carbon substrates (methanol + glycerol, methanol + sorbitol) at a predetermined "C-molar ratios" were added during the induction phases. Methanol- and sorbitol-based mixed feeding approach resulted in an improved huIFNα2b titer of 288 mg/L by channeling of methanol predominantly towards an optimal functioning of AOX expression system. A stand-off between biomass yield YXSand biomass heat yieldYQX coefficient, degree of reduction of methanol and its cosubstrate (glycerol and sorbitol) determines the fraction of carbon energy channeled toward biomass and protein production, under strict aerobic conditions. Calorespirometric monitoring and assessment of thermal yields enables a reliable prediction of process variables, leading to futuristic efficient PAT-based feed rate control.