Cell-free Enzyme System Research Articles

Preparation of a novel enzyme-modified cheddar cheese: Molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems

A novel enzyme-modified cheddar cheese was prepared and the molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems was investigated. By comparing five different protease-peptidase combinations, the group of neutral protease and flavor protease was found to increase the total leucine, valine, and isoleucine content (17.056 ± 0.136 g/kg) and the soluble nitrogen content was up to the level of a 12-month-matured cheese. Molecular docking and molecular dynamics simulations demonstrated their mode of action on four monomeric caseins. Adding a cell-free extract resulted in volatile flavor substances in the enzyme-modified cheese that were closest to those in the 12-month-matured cheese. This might be due to the flavor compensation effect of the conversion of leucine to 3-methylbutyraldehyde by transaminases and decarboxylases, and the conversion of 3-methylbutyric acid to 3-methylbutyraldehyde by ketoacid dehydrogenase and aldehyde dehydrogenase. This is essential for the enzyme modified cheddar cheese preparation.

Establishing a biochemical understanding of the initiation of chromosome replication in bacteria

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria. In a seminal paper in 1981, Fuller, Kaguni, and Kornberg reported the development of a cell-free enzyme system that could replicate DNA that was dependent on the bacterial origin of DNA replication, oriC. This advance opened the door to a flurry of discoveries and important papers that elucidated the process and control of initiation of chromosome replication in bacteria.

Integrating Carbohydrate and C1 Utilization for Chemicals Production.

In the face of increasing mobility and energy demand, as well as the mitigation of climate change, the development of sustainable and environmentally friendly alternatives to fossil fuels will be one of the most important tasks facing humankind in the coming years. In order to initiate the transition from a petroleum-based economy to a new, greener future, biofuels and synthetic fuels have great potential as they can be adapted to already common processes. Thereby, especially synthetic fuels from CO2 and renewable energies are seen as the next big step for a sustainable and ecological life. In our study, we directly address the sustainable production of the most common biofuel, ethanol, and the highly interesting next-generation biofuel, isobutanol, from methanol and xylose, which are directly derivable from CO2 and lignocellulosic waste streams, respectively, such integrating synthetic fuel and biofuel production. After enzyme and reaction optimization, we succeeded in producing either 3 g L-1 ethanol or 2 g L-1 isobutanol from 7.5 g L-1 xylose and 1.6 g L-1 methanol. In our cell-free enzyme system, C1-compounds are efficiently combined and fixed by the key enzyme transketolase and converted to the intermediate pyruvate. This opens the way for a hybrid production of biofuels, platform chemicals and fine chemicals from CO2 and lignocellulosic waste streams as alternative to conventional routes depending solely either on CO2 or sugars.

Cell‐Free Biosynthesis of ω‐Hydroxy Acids Boosted by a Synergistic Combination of Alcohol Dehydrogenases

The activity orchestration of an unprecedented cell-free enzyme system with self-sufficient cofactor recycling enables the stepwise transformation of aliphatic diols into ω-hydroxy acids at the expense of molecular oxygen as electron acceptor. The efficiency of the biosynthetic route was maximized when two compatible alcohol dehydrogenases were selected as specialist biocatalysts for each one of the oxidative steps required for the oxidative lactonization of diols. The cell-free system reached up to 100 % conversion using 100 mM of linear C5 diols and performed the desymmetrization of prochiral branched diols into the corresponding ω-hydroxy acids with an exquisite enantioselectivity (ee>99 %). Green metrics demonstrate superior sustainability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5-hydroxypetanoic acid. Finally, the cell-free system was assembled into a consortium of heterogeneous biocatalysts that allowed the enzyme reutilization. This cascade illustrates the potential of systems biocatalysis to access new heterofunctional molecules such as ω-hydroxy acids.

Extracts of Antrodia cinnamomea mycelium as a highly potent tyrosinase inhibitor.

Ganoderma has been known as a cure for diseases since ancient times, and been used as a medicinal mushroom for more than 2000 years. By many accounts, Ganoderma lucidum extracts from fruit bodies exhibited the comparable tyrosinase inhibition activity. To validate A. cinnamomea mycelia anti-melanogenesis activity. Ethanolic extracts of A. cinnamomea mycelia were evaluated using in vitro cell-free tyrosinase assay, cell-based and zebrafish phenotype-based method. Meanwhile, safety assessment was also conducted to ensure the feasibility as the novel ingredients in cosmetic and pharmaceutic industries. The major regulatory enzymes being in charge of cutaneous pigmentation, was investigated in both cell-free and cellular enzyme systems, and in phenotype-based zebrafish model. A high-throughput TLC in vitro screening system was introduced to perform the initial evaluation of those with anti-melanin formation activity. Among the fractions, 50% ethanol extracted fraction (AC_Et50_Hex) exhibited highest anti-melanin formation activity. AC_Et50_Hex (at 100 ppm) reduced 30% intracellular melanin of B16-F10 cells through suppression of tyrosinase activity and its protein expression. For animal study, not only does AC_Et50_Hex exhibited similar depigmenting efficacy to kojic acid (56.1% vs 52.3%) with lower dosage (50 ppm vs 1400 ppm), but showed less toxicity to zebrafish. A. cinnamomea mycelium extracts can be an ideal candidate/substitute for skin-whitening since kojic acid has been reported with carcinogenic effect. AC_Et50_Hex was recognized as a potential tyrosinase inhibitor throughout in vitro and in vivo analysis studies. The mass production of A. cinnamomea mycelium from agitated fermentation realizes the natural mushroom extracts for commercial application.

Click chemistry-based imaging to study the tissue distribution of the curcumin-protein complex in mice.

Previous studies have shown that curcumin, a bioactive dietary compound with a thiol-reactive α,β-unsaturated carbonyl moiety, can covalently modify protein thiols. However, most of the previous studies were performed in cultured cells or cell-free enzyme systems, and so it remains unknown whether curcumin could covalently modify proteins after oral administration in vivo. Using click chemistry-based fluorescence imaging, here we show that oral administration of dialkyne-curcumin (Di-Cur), a "click" probe mimicking curcumin, results in covalent modifications of cellular proteins in colon and liver tissues, but not in other tissues, in mice. This result suggests that oral administration of curcumin leads to the formation of the curcumin-protein complex in a tissue-specific manner, which could contribute to the biological effects and/or pharmacokinetics of curcumin. Further studies to elucidate the identities of curcumin-binding proteins could greatly help us to better understand the molecular mechanisms of curcumin, and develop novel strategies for disease prevention.

Enzymatic Bioremediation of Organophosphate Compounds-Progress and Remaining Challenges.

Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.

Self-assembly of bio-cellulose nanofibrils through intermediate phase in a cell-free enzyme system

The current study was aimed to investigate the synthesis and self-assembly of cellulose nanofibrils in in vitro environment through intermediate phase analysis and compare with the microbial cell system. The presence and stability of cellulose synthase and other essential enzymes were qualitatively validated through liquid chromatography–mass spectrometry/mass spectrometry linear trap quadrupole (LC–MS/MS LTQ) Orbitrap analysis of the culture medium after each batch (15 days’ point) of in vitro incubation. Monitoring of bio-cellulose synthesis in the in vitro environment revealed that β-1,4-glucan chains were de novo synthesized within the culture medium which crystallized and formed the premature bio-cellulose and bio-cellulose pellicles. The pellicle moved to the surface of the medium and ultimately formed a bio-cellulose sheet at the air-medium interface. In contrast to aerobic synthesis of cellulose (bacterial cellulose) by microbial cells, bio-cellulose was synthesized anaerboically by the cell-free enzyme system and the thickness of the sheet at the air-medium interface increased from the bottom surface facing the culture medium as confirmed by the field-emission scanning electron microscopy (FE-SEM). The current study will provide a base for in situ development of bionanocomposites of cellulose with various bactericidal and polymeric materials for broad spectrum applications.

Chemical profiling of edible seaweed (Ochrophyta) extracts and assessment of their in vitro effects on cell-free enzyme systems and on the viability of glutamate-injured SH-SY5Y cells

Neurodegenerative processes involve numerous and closely related events that ultimately culminate in neuronal cell injury. The aim of this study was (i) to assess, for the first time, the neuroprotective potential of acetone extracts of six edible species of Ochrophyta, by evaluating their cholinesterase and lipoxygenase inhibitory activity in cell-free assays, as well as their capacity to attenuate glutamate-induced toxicity in neuronal (SH-SY5Y) cells, and (ii) to try to relate the chemical composition of the extracts with their biological activity, evaluating also the effect of the main compounds thereof. In spite of a modest cholinesterase inhibition, a dose-dependent response towards lipoxygenase was found for all macroalgae extracts. At non-cytotoxic concentrations, the extracts from Fucus serratus Linnaeus and Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders were able to improve the viability of glutamate-insulted SH-SY5Y cells. These results encourage further studies for a more detailed understanding of the mechanisms beyond the documented biological activities, and point to the potential interest of the selected seaweed species and their extracts as promising candidates for in vivo studies.

Principles of Chemical Biology: APEX, Cell-Free Enzyme Systems, New Antibiotics, Epigenetic-Membrane Relationship, and Metastable Transcription

This month: GPCR signaling and networks in APEX, making terpenes via biocatalysis in a cell-free system, antibiotics for Gram-negative bugs, the role of membranes in epigenetics, and the role of metastability in transcription.

Recent advancements in bioreactions of cellular and cell-free systems: A study of bacterial cellulose as a model

Conventional approaches of regulating natural biochemical and biological processes are greatly hampered by the complexity of natural systems. Therefore, current biotechnological research is focused on improving biological systems and processes using advanced technologies such as genetic and metabolic engineering. These technologies, which employ principles of synthetic and systems biology, are greatly motivated by the diversity of living organisms to improve biological processes and allow the manipulation and reprogramming of target bioreactions and cellular systems. This review describes recent developments in cell biology, as well as genetic and metabolic engineering, and their role in enhancing biological processes. In particular, we illustrate recent advancements in genetic and metabolic engineering with respect to the production of bacterial cellulose (BC) using the model systems Gluconacetobacter xylinum and Gluconacetobacter hansenii. Besides, the cell-free enzyme system, representing the latest engineering strategies, has been comprehensively described. The content covered in the current review will lead readers to get an insight into developing novel metabolic pathways and engineering novel strains for enhanced production of BC and other bioproducts formation.

Single cell-strain부터 유래된 무세포 효소 시스템을 이용한 톨루엔 및 아세트산 분해

This study deals with the possible degradation of toluene and acetic acid when subjected to cell-free enzyme system from the toluene degrading bacteria Pseudomonas putida and acetic acid degrading bacteria Cupriavidus necator. P. putida produces toluene dioxygenase only under the existence of toluene in culture medium and toluene is degraded to cis-toluene dihydrodiol by this enzyme. C. necator produces acetyl coenzyme A synthetase-1 and converts acetic acid to acetyl CoA in order to synthesize ATP to need for growth or PHA which is biodegradable polymer. In case of toluene degradation, the experiment was conducted before and after production of toluene dioxygenase as this enzyme, produced by P. putida, is an inducible enzyme. Toluene was detected using gas chromatography (GC). Similar amount of toluene was found in control group and before production of toluene dioxygenase (experimental group 1). However, reduction in toluene was detected after the production of toluene dioxygenase (experimental group 2). Acetic acid was detected through application of gas chromatography-mass spectrometer (GC-MS). The results showed the acetic acid peak was not detected in the experimental group to apply cell-free enzyme system. These results show that the cell-free enzyme system obtained from P. putida and C. necator retained the ability to degrade toluene and acetic acid. However, P. putida needs to produce the inducible enzyme before preparation of the cell-free enzyme system.

Developmental strategies and regulation of cell-free enzyme system for ethanol production: a molecular prospective.

Most biomanufacturing systems developed for the production of biocommodities are based on whole-cell systems. However, with the advent of innovative technologies, the focus has shifted from whole-cell towards cell-free enzyme system. Since more than a century, researchers are using the cell-free extract containing the required enzymes and their respective cofactors in order to study the fundamental aspects of biological systems, particularly fermentation. Although yeast cell-free enzyme system is known since long ago, it is rarely been studied and characterized in detail. In this review, we hope to describe the major pitfalls encountered by whole-cell system and introduce possible solutions to them using cell-free enzyme systems. We have discussed the glycolytic and fermentative pathways and their regulation at both transcription and translational levels. Moreover, several strategies employed for development of cell-free enzyme system have been described with their potential merits and shortcomings associated with these developmental approaches. We also described in detail the various developmental approaches of synthetic cell-free enzyme system such as compartmentalization, metabolic channeling, protein fusion, and co-immobilization strategies. Additionally, we portrayed the novel cell-free enzyme technologies based on encapsulation and immobilization techniques and their development and commercialization. Through this review, we have presented the basics of cell-free enzyme system, the strategies involved in development and operation, and the advantages over conventional processes. Finally, we have addressed some potential directions for the future development and industrialization of cell-free enzyme system.

Bio-ethanol production through simultaneous saccharification and fermentation using an encapsulated reconstituted cell-free enzyme system

An encapsulated reconstituted cell-free enzyme system was developed through liquid-droplet forming method by using endogenous glycolytic and fermentation enzymes from yeast cells and exogenously added saccharification enzymes, cofactors, and ATPase. It was evaluated for bio-ethanol production through simultaneous saccharification and fermentation (SSF) at various temperatures, pH, and cell-free enzyme and substrate concentrations. Using 1% starch as substrate, encapsulated system illustrated maximum efficiency at 45°C and pH 7.0. SSF with encapsulated and bare reconstituted cell-free enzyme systems produced 3.47g/L and 2.98g/L bio-ethanol corresponding to 62% and 53% of maximum theoretical yield, respectively. It was explicable that encapsulated system provided better substrate utilization and product formation at elevated temperatures than bare system. Kinetic profile of SSF process of both systems was affected differently by variations in pH, temperature, and substrate and cell-free enzyme concentrations. Under appropriate conditions, system retained 90%, 64%, and 40% of initial enzyme concentration and produced 3.21, 2.24, and 0.83g/L bio-ethanol after 5, 10, and 15 consecutive batches, respectively. The current system offered several advantages and was superior compared to previously reported SSF systems. This system can effectively overcome the major barriers associated with successful development of SSF processes for bio-ethanol production on an industrial scale.

Yeast cell-free enzyme system for bio-ethanol production at elevated temperatures

A yeast cell-free enzyme system containing an intact fermentation assembly and that is capable of bio-ethanol production at elevated temperatures in the absence of living cells was developed to address the limitations associated with conventional fermentation processes. The presence of both yeast glycolytic and fermentation enzymes in the system was verified by SDS-PAGE and LC–MS/MS Q-TOF analyses. Quantitative measurements verified sufficient quantities of the co-factors ATP (1.8mM) and NAD+ (0.11mM) to initiate the fermentation process. Bio-ethanol was produced at a broad temperature range of 30–60°C but was highly specific to a pH range of 6.0–7.0. The final bio-ethanol production at 30, 40, 50, and 60°C was 3.37, 3.83, 1.94, and 1.60g/L, respectively, when a 1% glucose solution was used, and the yield increased significantly with increasing cell-free enzyme concentrations. A comparative study revealed better results for the conventional fermentation system (4.46g/L) at 30°C than the cell-free system (3.37g/L); however, the efficacy of the cell-free system increased with temperature, reaching a maximum (3.83g/L) at 40°C, at which the conventional system could only produce 0.48g/L bio-ethanol. Successful bio-ethanol production using a single yeast cell-based enzyme system at higher temperatures will lead to the development of novel strategies for efficient bio-ethanol production through SSF.

Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth

A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50°C in a citrate buffer system. The enzymes showed highest thermal stability at 30°C when exposed for prolonged time. As the temperature increased gradually from 25 to 70°C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70°C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentationwithout the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

Characteristics of the response of natural and recombinant luminescent microorganisms in the presence of Fe2+ ions

It was found that divalent iron ions have alternative effects on the bioluminescence of the natural marine microorganism Photobacterium phosphoreum and the recombinant Escherichia coli strain with a cloned lux operon of P. leiognathi. In the presence of 0.25-5.0 mM FeSO4, the bioluminescence intensity of the former and the latter increased and decreased, respectively. To establish the causes of these differences, we studied the characteristics of the fatty acid composition of the compared microorganisms. The fatty acid profile of E. coli was characterized by a high proportion of unsaturated 11-octadecenoic (vaccenic) acid. A study of this acid in a cell-free enzyme system used for bioluminescence generation showed that it is a potent inhibitor of bacterial bioluminescence. It was found that such effects are enhanced if 11-octadecenoid acid is preincubated with Fe2+.

Kinetics of the Oxidoreductase Involved in the Conversion of O‐methylsterigmatocystin to Aflatoxin B1

Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O‐methylsterigmatocystin (OMST) to the potent environmental carcinogen aflatoxin B1 (AFB1), has been proposed to be catalysed by an oxidoreductase (OR) that requires a cytochrome P‐450 type of oxidoreductase activity. This enzyme displays relative specificity towards OMST homologues in fungal whole cells. These studies were extended to the action of a cell‐free enzyme system (CFES), on five OMST homologues, with a view to establish the kinetics. In the current study a CFES, containing an oxidoreductase, was derived from a blocked mutant of Aspergillus parasiticus (Wh1‐11‐105). The key experimental steps involved rapid concentration and efficient dialysis by membrane filtration to remove small biomolecules (MW<10,000), co‐factors, primary and secondary metabolites. The kinetic parameters of the enzyme‐substrate reactions indicated that the reaction follows a Michealis‐Menten kinetics and OR activity decreased in the order: O‐butylsterigmatocystin>O‐propylsterigmatocystin>O‐ethylsterigmatocystin>O‐methylsterigmatocystin>O‐acetylsterigmatocystin>O‐benzoylsterigmatocystin. The 7‐O‐alkyl homologues were the best substrate for the CFES, thereby substantially supporting that the 7‐O‐methyl group of OMST is preferred for OR catalytic activity in the absence of any other alkylating groups in vitro. The Km was calculated as 5.65 µM for this CFES and varied marginally among the OMST homologues studied.

Preliminary assessment of the C13-side chain 2′-hydroxylase involved in Taxol biosynthesis

The biosynthesis of the anticancer drug Taxol in yew ( Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13- O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts α-phenylalanine to β-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield β-phenylalanoyl baccatin III (i.e., N-debenzoyl-2′-deoxytaxol) that undergoes subsequent 2′-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both β-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2′-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of β-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of β-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.

Myosin Light Chain Modification Depending on Magnetic Fields: II. Experimental

Recent experiments have revealed that Ca2+ -calmodulin dependent myosin light chain phosphorylation in a cell-free preparation exhibits unexpectedly high sensitivity to weak magnetic fields. This enzyme system is a well-studied biochemical system, which appears to depend upon ion binding. A previous article in this journal discussed the theoretical background of myosin phosphorylation and the ion-dependent interactions of EMF with soft tissues. Because of the electromagnetic field (EMF) sensitivity of this cell-free system, experiments were designed to test the effect of a pulsed radio frequency (PRF) field, pulsating magnetic fields (TEMF), gradient magnetic fields (Magnabloc), and homogeneous static magnetic fields (in Helmholtz arrangement) designed for clinical application. It is concluded that these various magnetic fields affect this cell-free enzyme system by modulating ion–protein interactions.