RESUMO
The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org .
Assuntos
Gerenciamento de Dados , Metadados , Reprodutibilidade dos Testes , Bases de Dados Factuais , CinéticaRESUMO
Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10 % salt solution was found to reduce tyrosine ammonia lyase activity by 19 %, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40 °C and pHâ 10 were found to be 3.8 % salt and 9.5 % polymer, and a 5 % potassium phosphate and 12.5 % poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for inâ situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33â g/L p-coumaric acid within 24â hours, a 1.9-fold improvement compared to biocatalysis without inâ situ product removal.
Assuntos
Amônia-Liases , Biocatálise , Ácidos Cumáricos , Propionatos , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Amônia-Liases/metabolismo , Amônia-Liases/química , Propionatos/química , Propionatos/metabolismo , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Fosfatos/metabolismo , Fosfatos/química , Propilenoglicóis/química , Propilenoglicóis/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Compostos de PotássioRESUMO
Advanced microbiome therapeutics have emerged as a powerful approach for the treatment of numerous diseases. While the genetic instability of genetically engineered microorganisms is a well-known challenge in the scale-up of biomanufacturing processes, it has not yet been investigated for advanced microbiome therapeutics. Here, the evolution of engineered Escherichia coli Nissle 1917 strains producing Interleukin 2 and Aldafermin were investigated in two strain backgrounds with and without the three error-prone DNA polymerases polB, dinB, and umuDC, which contribute to the mutation rate of the host strain. Whole genome short-read sequencing revealed the genetic instability of the pMUT-based production plasmid after serial passaging for approximately 150 generations using an automated platform for high-throughput microbial evolution in five independent lineages for six distinct strains. While a reduction of the number of mutations of 12%-43% could be observed after the deletion of the error-prone DNA polymerases, the interruption of production-relevant genes could not be prevented, highlighting the need for additional strategies to improve the stability of advanced microbiome therapeutics.
Assuntos
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Heterogeneidade GenéticaRESUMO
Bio-based production of fuels, chemicals and materials is needed to replace current fossil fuel based production. However, bio-based production processes are very costly, so the process needs to be as efficient as possible. Developments in synthetic biology tools has made it possible to dynamically modulate cellular metabolism during a fermentation. This can be used towards two-stage fermentations, where the process is separated into a growth and a production phase, leading to more efficient feedstock utilization and thus potentially lower costs. This article reviews the current status and some recent results in application of synthetic biology tools towards two-stage fermentations, and compares this approach to pre-existing ones, such as nutrient limitation and addition of toxins/inhibitors.
Assuntos
Fermentação , Engenharia Metabólica , Biologia Sintética , Engenharia Metabólica/métodosRESUMO
The group of natural aromatic compounds known as phenylpropanoids has diverse applications, but current methods of production which are largely based on synthesis from petrochemicals or extraction from agricultural biomass are unsustainable. Bioprocessing is a promising alternative, but improvements in production titers and rates are required to make this method profitable. Here the recent advances in genetic engineering and bioprocess concepts for the production of phenylpropanoids are presented for the purpose of identifying successful strategies, including adaptive laboratory evolution, enzyme engineering, in-situ product removal, and biocatalysis. The pros and cons of bacterial and yeast hosts for phenylpropanoid production are discussed, also in the context of different phenylpropanoid targets and bioprocess concepts. Finally, some broad recommendations are made regarding targets for continued improvement and areas requiring specific attention from researchers to further improve production titers and rates.
Assuntos
Engenharia Genética , Engenharia Metabólica , Biocatálise , Biomassa , BactériasRESUMO
Enzyme immobilization has been developing since the 1960s and although many industrial biocatalytic processes use the technology to improve enzyme performance, still today we are far from full exploitation of the field. One clear reason is that many evaluate immobilization based on only a few experiments that are not always well-designed. In contrast to many other reviews on the subject, here we highlight the pitfalls of using incorrectly designed immobilization protocols and explain why in many cases sub-optimal results are obtained. We also describe solutions to overcome these challenges and come to the conclusion that recent developments in material science, bioprocess engineering and protein science continue to open new opportunities for the future. In this way, enzyme immobilization, far from being a mature discipline, remains as a subject of high interest and where intense research is still necessary to take full advantage of the possibilities.
Assuntos
Enzimas Imobilizadas , Proteínas , Biocatálise , Engenharia , Enzimas Imobilizadas/metabolismo , Proteínas/metabolismoRESUMO
Enzymatic biodiesel production has been at the forefront of biofuels research in recent decades because of the significant environmental advantages it offers, while having the potential to be as effective as conventional chemically catalyzed biodiesel production. However, the higher capital cost, longer reaction time, and sensitivity of enzyme processes have restricted their widespread industrial adoption so far. It is also posited that the lack of research to bring the biodiesel product into final specification has scuppered industrial confidence in the viability of the enzymatic process. Furthermore, the vast majority of literature has focused on the development of immobilized enzyme processes, which seem too costly (and risky) to be used industrially. There has been little focus on liquid lipase formulations such as the Eversa Transform 2.0, which is in fact already used commercially for triglyceride transesterification. It is the objective of this review to highlight new research that focuses on bringing enzymatically produced biodiesel into specification via a liquid lipase polishing process, and the process considerations that come with it.
Assuntos
Biocombustíveis , Lipase , Biotecnologia , Enzimas Imobilizadas/metabolismo , Esterificação , Lipase/metabolismoRESUMO
Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.
Assuntos
Antibacterianos/biossíntese , Lisostafina/biossíntese , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Reatores Biológicos , Clonagem Molecular , Meios de Cultura/química , Meios de Cultura/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Glicerol/metabolismo , Glicerol/farmacologia , Lisostafina/genética , Lisostafina/isolamento & purificação , Lisostafina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus/genética , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme can improve its stability and enable its reuse multiple times, resulting in better performance and commercial viability. Consequently, biocatalysis is being widely applied in the production of pharmaceuticals and some commodity chemicals. Moreover, its broader application will be further stimulated in the future by the emerging biobased economy.
RESUMO
Despite enormous progress in protein engineering, complemented by bioprocess engineering, the revolution awaiting the application of biocatalysis in the fine chemical industry has still not been fully realized. In order to achieve that, further research is required on several topics, including (1) rapid methods for protein engineering using machine learning, (2) mathematical modelling of multi-enzyme cascade processes, (3) process standardization, (4) continuous process technology, (5) methods to identify improvements required to achieve industrial implementation, (6) downstream processing, (7) enzyme stability modelling and prediction, as well as (8) new reactor technology. In this brief mini-review, the status of each of these topics will be briefly discussed.
Assuntos
Biocatálise , Biotecnologia/métodos , Engenharia de Proteínas/métodos , Estabilidade Enzimática , Aprendizado de Máquina , Modelos TeóricosRESUMO
While the application of enzymes to synthetic and industrial problems continues to grow, the major development today is focused on multi-enzymatic cascades. Such systems are particularly attractive, because many commercially available enzymes operate under relatively similar operating conditions. This opens the possibility of one-pot operation with multiple enzymes in a single reactor. In this paper the concept of modules is introduced whereby groups of enzymes are combined in modules, each operating in a single reactor, but with the option of various operating strategies to avoid any complications of nonproductive interactions between the enzymes, substrates or products in a given reactor. In this paper the selection of modules is illustrated using the synthesis of the bulk chemical, gluconic acid, from lignocellulosic waste.
Assuntos
Catalase/química , Celulases/química , Gluconatos/síntese química , Glucose Oxidase/química , Lignina/química , Modelos Estatísticos , beta-Glucosidase/química , Biocatálise , Catalase/metabolismo , Celulases/metabolismo , Simulação por Computador , Fermentação , Gluconatos/química , Gluconatos/metabolismo , Glucose/química , Glucose/metabolismo , Glucose Oxidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lactonas/química , Lactonas/metabolismo , Lignina/metabolismo , Engenharia Metabólica/métodos , Temperatura , Resíduos , beta-Glucosidase/metabolismoRESUMO
As biocatalysis matures, it becomes increasingly important to establish methods with which to measure biocatalyst performance. Such measurements are important to assess immobilization strategies, different operating modes, and reactor configurations, aside from comparing protein engineered variants and benchmarking against economic targets. While conventional measurement techniques focus on a single performance metric (such as the total turnover number), here, it is argued that three metrics (achievable product concentration, productivity, and enzyme stability) are required for an accurate assessment of scalability.
Assuntos
Biocatálise , Enzimas Imobilizadas , Bioensaio/métodos , Estabilidade Enzimática , Cinética , TemperaturaRESUMO
As the application of biocatalysis to complement conventional chemical and catalytic approaches continues to expand, an increasing number of reactions involve poorly water-soluble substrates. At required industrial concentrations necessary for industrial implementation, this frequently leads to heterogeneous reaction mixtures composed of multiple phases. Such systems are challenging to sample, and therefore, it is problematic to measure representative component concentrations. Herein, an online method for following the progress of oxygen-dependent reactions through accurate measurement of the oxygen mass balance in the gas phase of a reactor is demonstrated and validated. The method was successfully validated and demonstrated by using two model reactions: firstly, the oxidation of glucose by glucose oxidase and, secondly, the Baeyer-Villiger oxidation of macrocyclic ketones to lactones. Initial reaction rate constants and time-course progressions calculated from the oxygen mass balance were validated against conventional online methods of dissolved oxygen tension and pH titration measurements. A feasible operating window and the sensitivity to dynamic changes of reaction rates were established by controlling oxygen transfer through the operating parameters of the reactor. Such kinetic data forms the basis for reaction characterisation, from which bottlenecks may be made evident and directed improvement strategies can be identified and implemented.
Assuntos
Glucose Oxidase/metabolismo , Oxigênio/metabolismo , Biocatálise , Concentração de Íons de Hidrogênio , Cetonas/química , Cetonas/metabolismo , Cinética , Lactonas/química , Lactonas/metabolismo , OxirreduçãoRESUMO
Thiol-ene (TE)-based polymer particles are traditionally prepared via emulsion polymerization in water (using surfactants, stabilizers, and cosolvents). Here, a green and simple alternative is presented with excellent control over particle size, while avoiding the addition of stabilizers. Glycerol is applied as a dispersing medium for the preparation of off-stoichiometric TE microparticles, where sizes in the range of 40-400 µm are obtained solely by changing the mixing speed of the emulsions prior to crosslinking. Control over surface chemistry is achieved by surface functionalization of excess thiol groups via photochemical thiol-ene chemistry resulting in a functional monolayer. In addition, surface chain transfer free radical polymerization is used for the first time to introduce a thicker polymer layer on the particle surface. The application potential of the system is demonstrated by using functional particles as adsorbent for metal ions and as a support for immobilized enzymes.
Assuntos
Glicerol/química , Compostos de Sulfidrila/síntese química , Radicais Livres/síntese química , Radicais Livres/química , Estrutura Molecular , Tamanho da Partícula , Processos Fotoquímicos , Polimerização , Polímeros/síntese química , Polímeros/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
Biocatalysis uses enzymes for chemical synthesis and production, offering selective, safe and sustainable catalysis. While today the majority of applications are in the pharmaceutical sector, new opportunities are arising every day in other industry sectors, where production costs become a more important driver. In the early applications of the technology, it was necessary to design processes to match the properties of the biocatalyst. With the advent of protein engineering, organic chemists started to develop and improve enzymes to suit their needs. Likewise in industry, although not widespread, a new paradigm was already implemented several years ago to engineer enzymes to suit process needs. Today, a new era is entered, where the effectiveness with which such integrated protein and process engineering is achieved becomes critical to implementation. In this paper, the development of a tool to improve the effectiveness of this approach is discussed, namely the use of target-setting based on process requirements, to guide the necessary protein engineering.This article is part of a discussion meeting issue 'Providing sustainable catalytic solutions for a rapidly changing world'.
RESUMO
Biocatalytic oxidation reactions employing molecular oxygen as the electron acceptor are difficult to conduct in a continuous flow reactor because of the requirement for high oxygen transfer rates. In this paper, the oxidation of glucose to glucono-1,5-lactone by glucose oxidase was used as a model reaction to study a novel continuous agitated cell reactor (ACR). The ACR consists of ten cells interconnected by small channels. An agitator is placed in each cell, which mixes the content of the cell when the reactor body is shaken by lateral movement. Based on tracer experiments, a hydrodynamic model for the ACR was developed. The model consisted of ten tanks-in-series with back-mixing occurring within and between each cell. The back-mixing was a necessary addition to the model in order to explain the observed phenomenon that the ACR behaved as two continuous stirred tank reactors (CSTRs) at low flow rates, while it at high flow rates behaved as the expected ten CSTRs in series. The performance of the ACR was evaluated by comparing the steady state conversion at varying residence times with the conversion observed in a stirred batch reactor of comparable size. It was found that the ACR could more than double the overall reaction rate, which was solely due to an increased oxygen transfer rate in the ACR caused by the intense mixing as a result of the spring agitators. The volumetric oxygen transfer coefficient, kL a, was estimated to be 344 h-1 in the 100 mL ACR, opposed to only 104 h-1 in a batch reactor of comparable working volume. Interestingly, the large deviation from plug flow behavior seen in the tracer experiments was found to have little influence on the conversion in the ACR, since both a plug flow reactor (PFR) model and the backflow cell model described the data sufficiently well. Biotechnol. Bioeng. 2017;114: 1222-1230. © 2017 Wiley Periodicals, Inc.
Assuntos
Reatores Biológicos , Glucose Oxidase/química , Glucose/química , Modelos Químicos , Oxigênio/química , Reologia/instrumentação , Técnicas de Cultura Celular por Lotes/instrumentação , Catálise , Simulação por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Gluconatos/síntese química , Lactonas/síntese química , OxirreduçãoRESUMO
An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid-liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L-1 , a purity up to 70% gMPPA · gtot-1 and a recovery in the range of 80% mol · mol-1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600-609. © 2016 Wiley Periodicals, Inc.
Assuntos
Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Reatores Biológicos , Técnicas de Cultura Celular por Lotes , Biocatálise , Produtos Biológicos/química , Biotecnologia , Enzimas Imobilizadas/metabolismo , Microbiologia Industrial , Modelos Biológicos , Propilaminas/análise , Propilaminas/química , Propilaminas/isolamento & purificação , Propilaminas/metabolismo , Estereoisomerismo , Transaminases/metabolismoRESUMO
The alkaline process for making biodiesel (fatty acid methyl esters, or FAME) is highly efficient at the transesterification of glycerides. However, its performance is poor when it comes to using oil that contain significant amounts of free fatty acids (FFA). The traditional approach to such feedstocks is to employ acid catalysis, which is slow and requires a large excess of methanol, or to evaporate FFA and convert that in a separate process. An attractive option would be to convert the FFA in oil feedstocks to FAME, before introducing it into the alkaline process. The high selectivity of enzyme catalysis makes it a suitable basis for such a pretreatment process. In this work, we present a characterization of the pretreatment of high-FFA rapeseed oil using immobilized Candida antarctica lipase B (Novozym 435), focused on the impact of initial FFA and methanol concentration. Based on experimental results, we have identified limitations for the process in terms of FFA concentration in the feedstock and make suggestions for process operation. It was found that, using 5% catalyst and 4% methanol at 35°C, the FFA concentration could be reduced to 0.5% within an hour for feedstock containing up to 15% FFA. Further, the reaction was observed to be under kinetic control, in that the biocatalyst converts FFA (and FAME) at a much higher rate than glyceride substrates. There is thus, both a minimum and a maximum reaction time for the process to achieve the desired concentration of FFA. Finally, an assessment of process stability in a continuous packed bed system indicates that as much as 15 m(3) oil could potentially be pretreated by 1 kg of biocatalyst at the given process conditions.
Assuntos
Biocombustíveis , Ácidos Graxos/metabolismo , Lipase/metabolismo , Metanol/metabolismo , Óleos de Plantas/metabolismo , Biotransformação , Enzimas Imobilizadas/metabolismo , Ácidos Graxos/análise , Ácidos Graxos Monoinsaturados , Proteínas Fúngicas , Cinética , Óleo de Brassica napus , TemperaturaRESUMO
In this work, we demonstrate the scale-up from an 80 L fed-batch scale to 40 m(3) along with the design of a 4 m(3) continuous process for enzymatic biodiesel production catalyzed by NS-40116 (a liquid formulation of a modified Thermomyces lanuginosus lipase). Based on the analysis of actual pilot plant data for the transesterification of used cooking oil and brown grease, we propose a method applying first order integral analysis to fed-batch data based on either the bound glycerol or free fatty acid content in the oil. This method greatly simplifies the modeling process and gives an indication of the effect of mixing at the various scales (80 L to 40 m(3) ) along with the prediction of the residence time needed to reach a desired conversion in a CSTR. Suitable process metrics reflecting commercial performance such as the reaction time, enzyme efficiency, and reactor productivity were evaluated for both the fed-batch and CSTR cases. Given similar operating conditions, the CSTR operation on average, has a reaction time which is 1.3 times greater than the fed-batch operation. We also showed how the process metrics can be used to quickly estimate the selling price of the enzyme. Assuming a biodiesel selling price of 0.6 USD/kg and a one-time use of the enzyme (0.1% (w/woil ) enzyme dosage); the enzyme can then be sold for 30 USD/kg which ensures that that the enzyme cost is not more than 5% of the biodiesel revenue. Biotechnol. Bioeng. 2016;113: 1719-1728. © 2016 Wiley Periodicals, Inc.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Biocombustíveis , Reatores Biológicos , Lipase/metabolismo , Eurotiales/enzimologia , Microbiologia IndustrialRESUMO
We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.