Enzymatic Cascade Reaction Research Articles

NEDD4 and NEDD4L: Ubiquitin Ligases Closely Related to Digestive Diseases.

Protein ubiquitination is an enzymatic cascade reaction and serves as an important protein post-translational modification (PTM) that is involved in the vast majority of cellular life activities. The key enzyme in the ubiquitination process is E3 ubiquitin ligase (E3), which catalyzes the binding of ubiquitin (Ub) to the protein substrate and influences substrate specificity. In recent years, the relationship between the subfamily of neuron-expressed developmental downregulation 4 (NEDD4), which belongs to the E3 ligase system, and digestive diseases has drawn widespread attention. Numerous studies have shown that NEDD4 and NEDD4L of the NEDD4 family can regulate the digestive function, as well as a series of related physiological and pathological processes, by controlling the subsequent degradation of proteins such as PTEN, c-Myc, and P21, along with substrate ubiquitination. In this article, we reviewed the appropriate functions of NEDD4 and NEDD4L in digestive diseases including cell proliferation, invasion, metastasis, chemotherapeutic drug resistance, and multiple signaling pathways, based on the currently available research evidence for the purpose of providing new ideas for the prevention and treatment of digestive diseases.

Ultrasmall copper nanodots eye drops for microenvironment regulation of diabetes retinopathy by relieving hypoxia and inhibiting HIF-1α/VEGF signaling pathway

Diabetic retinopathy (DR) is the main cause of visual loss among blinding diseases. The pathological microenvironment, characterized by hypoxia, reactive oxygen species (ROS) accumulation and oxidative stress, is critical to the onset and progression of DR. In this study, an ultrasmall (4–6 nm) copper nanodots was developed as nanozyme (Cu NZs) and dispersed in 2 % Methocel to formulate Cu NZs eye drops to relieve hypoxia and scavenge excrescent ROS for the effective treatment of DR. Due to its smaller size and higher specific surface area, Cu NZs exposes more active sites and can guarantee excellent antioxidant activity. The results of in vitro studies indicated that Cu NZs had biocompatible and protective effects on human retinal microvascular endothelial cells (HRMECs), alleviating hypoxia in HRMECs, reducing oxidative stress, and inhibiting hypoxia-inducing factor-1α (HIF-1α)/ vascular endotheliogenesis factor (VEGF) signaling pathway. In an animal model of streptozotocin (STZ)-induced DR, Cu NZs eye drops have been shown to greatly relieve the hypoxic microenvironment of DR and inhibit the expression of HIF-1α and VEGF. CuNZs eye drops reduced inflammatory factors in the DR microenvironment, significantly reducing the progression of neovascularization and vascular leakage. And the nano-enzyme eye drops have no toxic side effects on the eyes and important organs of the whole body. This treatment mode based on the nano enzyme cascade reaction from comprehensive microenvironment regulation (hypoxia relief, free radical scavenging, anti-inflammatory), has excellent ability to treat DR. Although the nanoparticle is effective against DR in the short term at the cellular and animal levels, the long-term efficacy and safety remains to be evaluated.

Development and characterization of a bacterial enzyme cascade reaction system for efficient and stable PET degradation

The widespread use of polyethylene terephthalate (PET) in various industries has led to a surge in microplastics (MPs) pollution, posing a significant threat to ecosystems and human health. To address this, we have developed a bacterial enzyme cascade reaction system (BECRS) that focuses on the efficient degradation of PET. This system harnesses the Escherichia coli (E. coli) surface to display CsgA protein, which forms curli fibers, along with the carbohydrate-binding module 3 (CBM3) and PETases, to enhance the adsorption and degradation of PET. The study demonstrated that the BECRS achieved a notable PET film degradation rate of 3437 ± 148 μg/(d*cm²), with a degradation efficiency of 21.40% for crystalline PET MPs, and the degradation products were all converted to TPA. The stability of the system was evidenced by retaining over 80% of its original activity after multiple uses and during one month of storage. Molecular dynamics simulations confirmed that the presence of CsgA did not interfere with the enzymatic activity of PETases. This BECRS represents a significant step forward in the biodegradation of PET, particularly microplastics, offering a practical and sustainable solution for environmental pollution control.

Modeling Enzymatic Cascade Reactions Immobilized in Plug‐Flow Reactors for Flow Biocatalysis

AbstractFlow biocatalysis has emerged as a promising technology for the sustainable production of chemicals. Multiple immobilized enzymes are often used as biocatalysts to perform enzymatic cascade reactions in flow biocatalytic systems. When designing packed‐bed reactors with multiple immobilized enzymes, the enzymes are distributed in porous particles, and the porous particles are distributed in catalyst zones. A recently developed methodology is extended for selecting spatial immobilization distributions in batch reactors to packed‐bed reactors. Mechanistic models are used to compare the three most basic designs for a packed‐bed reactor, i.e., mixed individually immobilized enzymes, separated individually immobilized enzymes, and mixed co‐immobilized enzymes. For first‐order microkinetics, the design with co‐immobilized enzymes was found to always outperform the other two. Separating individually immobilized enzymes in two zones was proven to outperform mixing them in one zone for any positive order reaction kinetic.

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Distance-based paper sensor for DNase I detection based on aptamer-mediated enzyme cascade reaction

DNase I is proven to be an early predictive molecule for a variety of diseases, which is important for early diagnosis and clinical detection of diseases. Herein, we demonstrate a distance-based paper sensor to detect DNase I via an enzymatic cascade reaction mediated by the thrombin aptamer. In the presence of DNase I, the thrombin is able to clot the plasma normally because the aptamer chain is hydrolyzed by DNase I and cannot bind thrombin properly to inhibit thrombin activity. The concentration of DNase I is negatively correlated with the volume of mobile phase in sample solution, and it can be quantified by measuring the flow distance of water on the pH indicator strip. The distance-based paper sensor has good specificity, and the detection limit is 2 × 10−2 U/mL. In addition, it can be used for DNase I analysis in human serum samples to show its efficacy in the early diagnosis of diseases associated with DNase I activity. Overall, we developed a simple, fast and low-cost DNase I detection method, which has great commercial potential.

Injectable Hydrogel Based on Enzymatic Initiation of Keratin Methacrylate for Controlled Exosome Release in Intervertebral Disc Degeneration Therapy

AbstractThe treatment of intervertebral disc degeneration (IVDD) using bone marrow mesenchymal stem cell‐derived exosomes has shown success in alleviating inflammation and restoring the extracellular matrix (ECM), however, challenges persist due to the deficiency in mechanical support and controlled release. Herein, a carbon‐carbon double bond modified keratin (KeMA) is synthesized by 2‐isocyanatoethyl modification for exosomes wrapping. This injectable KeMA hydrogel, initiated by a biocompatible glucose/ glucose oxidase/ horse radish peroxidase enzymatic cascade reaction with acetylacetone and N‐vinylpyrrolidone, displayed rapid gelation, resembling nucleus pulposus (NP) elasticity, and excellent cytocompatibility. In vitro studies showcased that the exosomes‐loaded KeMA hydrogel (Exo@KeMA) enhanced exosome release kinetics, suppressed inflammation, fostered extracellular matrix (ECM) regeneration, and reinstated NP biomechanics. RNA‐seq analysis indicated Exo@KeMA's effects involved PI3K‐Akt signaling for matrix regeneration and NF‐κB signaling inhibition for anti‐inflammation. In vivo IVDD rat models demonstrated Exo@KeMA attenuated inflammation, maintained NP water content, preserved disc height, and promoted structural regeneration. This research introduces an injectable KeMA hydrogel as a promising therapy for IVDD, by facilitating biomechanics restoration, anti‐inflammatory response, and ECM regeneration.

Rapid and Cost-Effective On-site Detection of Plant Viruses Using Personal Glucose Meters Integrated with LAMP and Cascade Enzymatic Reactions

Rapid and Cost-Effective On-site Detection of Plant Viruses Using Personal Glucose Meters Integrated with LAMP and Cascade Enzymatic Reactions

Whole-cell catalytic production of pseudouridine by recombinant Escherichia coli

Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli, enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.

Semiconducting polymer dots based l-lactate sensor by enzymatic cascade reaction system

Backgroundl-lactate detection is important for not only assessing exercise intensity, optimizing training regimens, and identifying the lactate threshold in athletes, but also for diagnosing conditions like L-lactateosis, monitoring tissue hypoxia, and guiding critical care decisions. Moreover, l-lactate has been utilized as a biomarker to represent the state of human health. However, the sensitivity of the present l-lactate detection technique is inadequate. ResultsHere, we reported a sensitive ratiometric fluorescent probe for l-lactate detection based on platinum octaethylporphyrin (PtOEP) doped semiconducting polymer dots (Pdots-Pt) with enzymatic cascade reaction. With the help of an enzyme cascade reaction, the l-lactate was continuously oxidized to pyruvic and then reduced back to l-lactate for the next cycle. During this process, oxygen and NADH were continuously consumed, which increased the red fluorescence of Pdots-Pt that responded to the changes of oxygen concentration and decreased the blue fluorescence of NADH at the same time. By comparing the fluorescence intensities at these two different wavelengths, the concentration of l-lactate was accurately measured. With the optimal conditions, the probes showed two linear detection ranges from 0.5 nM to 5.0 μM and 5.0 μM–50.0 μM for l-lactate detection. The limit of detection was calculated to be 0.18 nM by 3σ/slope method. Finally, the method shows good detection performance of l-lactate in both bovine serum and artificial serum samples, indicating its potential usage for the selective analysis of l-lactate for health monitoring and disease diagnosis. SignificanceThe successful application of the sensing system in the complex biological sample (bovine serum and artificial serum samples) demonstrated that this method could be used for sensitive l-lactate detection in practical clinical applications. This detection system provided an extremely low detection limit, which was several orders of magnitude lower than methods proposed in other literatures.

Acetylcholine triggered enzymatic cascade reaction based on Fe7S8 nanoflakes catalysis for organophosphorus pesticides visual detection

BackgroundOrganophosphorus pesticides (OPs) play important roles in the natural environment, agricultural fields, and biological prevention. The development of OPs detection has gradually become an effective strategy to avoid the dangers of pesticides abuse and solve the severe environmental and health problems in humans. Although conventional assays for OPs analysis such as the bulky instrument required analytical methods have been well-developed, it still remains the limitation of inconvenient, inefficient and lab-dependence analysis in real samples. Hence, there is an urgent demand to develop efficient detection methods for OPs analysis in real scenarios. ResultsHere, by virtue of the highly efficient catalytic performance in Fe7S8 nanoflakes (Fe7S8 NFs), we propose an OPs detection method that rationally integrated Fe7S8 NFs into the acetylcholine (ACh) triggered enzymatic cascade reaction (ATECR) for proceeding better detection performances. In this method, OPs serve as the enzyme inhibitors for inhibiting ATECR among ACh, acetylcholinesterase (AChE), and choline oxidase (CHO), then reduce the generation of H2O2 to suppress the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) that catalyzed by Fe7S8 NFs. Benefiting from the integration of Fe7S8 NFs and ATECR, it enables a sensitive detection for OPs (e.g. dimethoate). The proposed method has presented good linear ranges of OPs detection ranging from 0.1 to 10 μg mL−1. Compared to the other methods, the comparable limits of detection (LOD) of OPs are as low as 0.05 μg mL−1. SignificanceFurthermore, the proposed method has also achieved a favorable visual detection performance of revealing OPs analysis in real samples. The visual signals of OPs can be transformed into RGB values and gathered by using smartphones, indicating the great potential in simple, sensitive, instrument-free and on-site analysis of pesticide residues in environmental monitoring and biosecurity research.

Modeling enzymatic and electrochemical cascade reactions at the three‐phase interface enzyme electrode

AbstractThe enzyme electrode based on enzymatic and electrochemical cascade reactions is a golden approach for detecting various biomarkers. How the interfacial architectures of a promising three‐phase interface enzyme electrode can influence the cascade reactions and electrode performance, however, remains unclear. In this study, a mathematical model has been developed to describe intraphase and interphase mass transfer, coupled with cascade reactions. The results reveal that the interfacial architectures determine the mass transfer of oxygen (substrate of oxidase) and H2O2 (enzymatic product) in the cascade reactions, hence affecting the electrode current. Generally, thinner pore walls facilitate the mass transfer of oxygen, thereby enhancing the enzymatic kinetics and H2O2 production. Meanwhile, smaller pore sizes shorten the diffusion pathway of H2O2 to the electrode surface, thereby increasing the electrocatalytic reaction rate. This work provides an efficient in silico tool for the design of high‐performance three‐phase enzyme electrodes.

Construction of Double-enzyme Complexes with DNA Framework Nanorulers for Improving Enzyme Cascade Catalytic Efficiency.

Efficient biocatalytic cascade reactions play a crucial role in guiding intricate, specific and selective intracellular transformation processes. However, the catalytic activity of the enzyme cascade reaction in bulk solution was greatly impacted by the spatial morphology and inter-enzyme distance. The programmability and addressability nature of framework nucleic acid (FNA) allows to be used as scaffold for immobilization and to direct the spatial arrangement of enzyme cascade molecules. Here, we used tetrahedral DNA framework (TDF) as nanorulers to assemble two enzymes for constructing a double-enzyme complex, which significantly enhance the catalytic efficiency of sarcosine oxidase (SOx)/horseradish peroxidase (HRP) cascade system. We synthesized four types of TDF nanorulers capable of programming the lateral distance between enzymes from 5.67 nm to 12.33 nm. Enzymes were chemical modified by ssDNA while preserving most catalytic activity. Polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to verify the formation of double-enzyme complex. Four types of double-enzyme complexes with different enzyme distance were constructed, in which TDF26(SOx+HRP) exhibited the highest relative enzyme cascade catalytic activity, ~3.11-fold of free-state enzyme. Importantly, all the double-enzyme complexes demonstrate a substantial improvement in enzyme cascade catalytic activity compared to free enzymes.

One-Pot Synthesis of Useful S-Substituted-l-cysteine Sulfoxides Using Genetically Engineered Escherichia coli.

S-Substituted-l-cysteine sulfoxides are valuable compounds that are contained in plants. Particularly, (+)-alliin and its degraded products have gained significant attention because of their human health benefits. However, (+)-alliin production has been limited to extraction from plants and chemical synthesis; both methods have drawbacks in terms of stability and safety. Here, we proposed the enzymatic cascade reaction for synthesizing (+)-alliin from readily available substrates. To achieve a one-pot (+)-alliin production, we constructed Escherichia coli coexpressing the genes encoding tryptophan synthase from Aeromonas hydrophila ssp. hydrophila NBRC 3820 and l-isoleucine hydroxylase from Bacillus thuringiensis 2e2 for the biocatalyst. Deletion of tryptophanase gene in E. coli increased the yield about 2-fold. Under optimized conditions, (+)-alliin accumulation reached 110 mM, which is the highest productivity thus far. Moreover, natural and unnatural S-substituted-l-cysteine sulfoxides were synthesized by applying various thiols to the cascade reaction. These results indicate that the developed bioprocess would enable the supply of diverse S-substituted-l-cysteine sulfoxides.

Paper-based ligand fishing method for rapid screening and real-time capturing of α-glucosidase inhibitors from the Chinese herbs

Identifying medicinally relevant compounds from natural resources generally involves the tedious work of screening plants for the desired activity before capturing the bioactive molecules from them. In this work, we created a paper-based ligand fishing platform to vastly simplify the discovery process. This paper-based method exploits the enzymatic cascade reaction between α-glucosidase (GAA), glucose oxidase (GOx), and horseradish peroxidase (HRP), to simultaneously screen the plants and capture the GAA inhibitors from them. The designed test strip could capture ligands in tandem with screening the plants, and it features a very simply operation based on direct visual assessment. Multiple acylated flavonol glycosides from the leaves of Quercus variabilis Blume were newly found to possess GAA inhibitory activities, and they may be potential leads for new antidiabetic medications. Our study demonstrates the prospect of the newly discovered GAA ligands as potential bioactive ingredients as well as the utility of the paper-based ligand fishing method.

Acid‐Unlocked Switch Controlled the Enzyme and CO In Situ Release to Induce Mitochondrial Damage via Synergy

AbstractCO gas therapy has attracted enormous attention in tumor therapy due to the abilities of mitochondrial damage and inhibition of cellular respiration. However, the inefficient and random release of CO greatly limit its application. Taking this into account, the study constructs an acid‐unlocked nanostructure based on MPDA‐MnCO‐GOx@DSNPs, designated as MMGD. The nanostructure enables tumor microenvironment (TME) specific enzyme and CO prodrug (manganese carbonyl, MnCO) cascade reaction, thus facilitating CO release in situ. Mesoporous polydopamine (MPDA) can provide the space for MnCO and glucose oxidase (GOx) loading. Especially, lanthanide (Ln3+)‐doped down‐shifting luminescent nanoparticles (DSNPs) can not only serve as the near‐infrared II (NIR‐II) fluorescence imaging probe, but also act as the acid‐unlocked gating switch. The slightly acidic TME can render the dissociation of DSNPs, thus exposing GOx and releasing MnCO. The catalytic reaction of GOx can produce H2O2 and create a more acidic environment, which facilitates the CO generation in situ, leading to mitochondrial damage by reducing cytochrome c oxidase activity and adenosine triphosphate (ATP) levels. Meanwhile, MPDA has the NIR light absorption capability for photothermal therapy (PTT). This study provides an ingenious strategy for efficient and controllable CO gas, starvation, and PTT of tumor guided by NIR‐II fluorescence imaging.

Protein engineering of transaminase facilitating enzyme cascade reaction for the biosynthesis of azasugars

Azasugars, such as 1-deoxynojirimycin (1-DNJ), exhibit unique physiological functions and hold promising applications in medicine and health fields. However, the biosynthesis of 1-DNJ is hindered by the low activity and thermostability of the transaminase. In this study, the transaminase from Mycobacterium vanbaalenii (MvTA) with activity toward d-fructose was engineered through semi-rational design and high-throughput screening method. The final mutant M9-1 demonstrated a remarkable 31.2-fold increase in specific activity and an impressive 200-fold improvement in thermostability compared to the wild-type enzyme. Molecular dynamics (MD) simulations revealed that the mutation sites of H69R and K145R in M9-1 played crucial roles in the binding of the amino acceptor and donor, leading to the stable conformation of substrates within the active pocket. An enzyme cascade reaction was developed using M9-1 and the dehydrogenase from Paenibacillus polymyxa (GutB1) for the production of mannojirimycin (MJ), which provided a new idea for the invitro biosynthesis of 1-DNJ.

Reusable Colorimetric Biosensors on Sustainable Silk-Based Platforms.

In biosensor development, silk fibroin is advantageous for providing transparent, flexible, chemically/mechanically stable, biocompatible, and sustainable substrates, where the biorecognition element remains functional for long time periods. These properties are employed here in the production of point-of-care biosensors for resource-limited regions, which are able to display glucose levels without the need for external instrumentation. These biosensors are produced by photopatterning silk films doped with the enzymes glucose oxidase and peroxidase and photoelectrochromic molecules from the dithienylethene family acting as colorimetric mediators of the enzymatic reaction. The photopatterning results from the photoisomerization of dithienylethene molecules in the silk film from its initial uncolored opened form to its pink closed one. The photoisomerization is dose-dependent, and colored patterns with increasing color intensities are obtained by increasing either the irradiation time or the light intensity. In the presence of glucose, the enzymatic cascade reaction is activated, and peroxidase selectively returns closed dithienylethene molecules to their initial uncolored state. Color disappearance in the silk film is proportional to glucose concentration and used to distinguish between hypoglycemic (below 4 mM), normoglycemic (4-6 mM), and hyperglycemic levels (above 6 mM) by visual inspection. After the measurement, the biosensor can be regenerated by irradiation with UV light, enabling up to five measurement cycles. The coupling of peroxidase activity to other oxidoreductases opens the possibility to produce long-life reusable smart biosensors for other analytes such as lactate, cholesterol, or ethanol.

Multienzyme cascades analysis of α-glucosidase by oxygen deficient MoO3-x

BackgroundRecently, the enzymatic cascade reactions during the cellular process are widely used for fabricating robust biosensors and they have attracted extensive attention in analyzing various clinical biomarkers. The enzymatic cascades analysis is commonly based on the peroxidase (POD)/oxidase coupled system. However, the requirement of harsh acidic environment, poor stability and interference from the oxidase further limit their analytical practicability. Herein, novel chromogenic nanomaterials with H2O2 sensitive features are urgently required to replace the POD nanozyme in enzymatic cascades based bioanalysis. ResultsHerein, oxygen deficient MoO3-x with H2O2 sensitive features and near-infrared (NIR) absorption band have been ultra-fast synthesized and utilized for the enzymatic cascades analysis of α-Glucosidase's activity, and inhibitors screening. With the addition of 4-nitrophenyl-α-d-glucopyranoside, the simultaneous presence of α-Glucosidase and glucose oxidase (GOx) would fade their dark blue color and decrease the NIR absorption. The α-Glucosidase's activity can be analyzed by the absorption at 770 nm, and their limit of detection is 8 × 10−5 U/mL, indicating the superior performance of the proposed colorimetric assay. Moreover, this proposed α-Glucosidase assay is further utilized for inhibitors screening. Moreover, the activity of α-Glucosidase can also be analyzed by the smartphone and microplate reader through the agarose-based colorimetric portable kit. SignificanceThis MoO3-x involved enzymatic cascades assay would facilitate for the development of bio-analysis related to H2O2 generation or consumption. Moreover, this bio-analysis strategy will contribute to the development of other H2O2 sensitive chromogenic nanomaterials for the analysis of certain biomolecules and biological enzymes.

Dual enzymatic preparation of flexible biocomplex with full amino-acid composition

The preparation of highly versatile flexible biomaterials able to adapt to the complex environment of the human body is an important but challenging task. In this study, a flexible biocomplex (AA-FB) was prepared by combining gelatin, polylysine, and skin using a green enzymatic cascade reaction. Interestingly, none of the raw materials used in the preparation of AA-FB introduced any compounds other than amino-acid composition, which can endow AA-FB with low biotoxicity and allergenicity, along with excellent biodegradability. Morever, AA-FB showed good physical properties: its Young's modulus was 30 times higher than that of pure gelatin hydrogel, and its water vapor transmission rate (2480.12 g/m2·24 h) was also significantly improved. AA-FB could also achieve a bacterial inhibition rate of 91.34 % against S. aureus, much higher than that of skin (28.51 %) and gelatin (41.89 %). Tests of artificial skin show that AA-FB can replace skin tissue as a protective barrier for collagen, blood vessels, and cells. As the new skin tissue is reconstructed, it gradually integrates with the normal tissue of the body until is finally metabolized.