Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) as a compensatory protease inhibitor in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Photoelectrochemical Solution Gated Graphene Field-Effect Transistor Functionalized by Enzymatic Cascade Reaction for Organophosphate Detection.

Solution Gated Graphene Field-Effect Transistors (SGGT) are eagerly anticipated as an amplification platform for fabricating advanced ultra-sensitive sensors, allowing significant modulation of the drain current with minimal gate voltage. However, few studies have focused on light-matter interplay gating control for SGGT. Herein, this challenge is addressed by creating an innovative photoelectrochemical solution-gated graphene field-effect transistor (PEC-SGGT) functionalized with enzyme cascade reactions (ECR) for Organophosphorus (OPs) detection. The ECR system, consisting of acetylcholinesterase (AChE) and CuBTC nanomimetic enzymes, selectively recognizes OPs and forms o-phenylenediamine (oPD) oligomers sediment on the PEC electrode, with layer thickness related to the OPs concentration, demonstrating time-integrated amplification. Under light stimulation, the additional photovoltage generated on the PEC gate electrode is influenced by the oPD oligomers sediment layer, creating a differentiated voltage distribution along the gate path. PEC-SGGT, inherently equipped with built-in amplification circuits, sensitively captures gate voltage changes and delivers output with an impressive thousandfold current gain. The seamless integration of these three amplification modes in this advanced sensor allows a good linear range and highly sensitive detection of OPs, with a detection limit as low as 0.05 pm. This work provides a proof-of-concept for the feasibility of light-assisted functionalized gate-controlled PEC-SGGT for small molecule detection.

Investigation on production and reaction conditions of sucrose synthase based glucosylation cascade towards flavonoid modification.

Enzyme-based glycosylation is of great interest in the context of natural products decoration. Yet, its industrial application is hindered by optimisation difficulties and hard-to-standardise productivities. In this study, five sugar nucleotide-dependent glucosyltransferases from different origins (bacterial, plant and fungal) were coupled with soy sucrose synthase (GmSuSy) to create a set of diverse cascade biocatalysts for flavonoid glucosylation, which evaluation brought new insights into the field. Investigations into co-expression conditions and reaction settings enabled to define optimal induction temperature (25 °C) and uridine diphosphate (UDP) concentration (0.5 mM) for all tested pairs of enzymes. Moreover, the influence of pH and substrate concentration on the monoglucosylated product distribution was detected and analysed. The utilisation of crude protein extracts as a cost-effective source of catalysts unveiled their glycosidase activity against flavonoid glucosides, resulting in decreased productivity, which, to our knowledge, has not previously been discussed in such a context. Additionally, examination of the commercially available EziG immobilisation resins showed that selection of suitable carrier for solid catalyst production can be problematic and not only enzyme's but also reagent's properties have to be considered. Flavonoids, due to their complexation and hydrophobic properties, can adsorb on different types of surfaces, including divalent metal ions required for IMAC based immobilisation, necessitating detailed examination of the resins while the catalysis design.

Enzymatic biosynthesis of D-galactose derivatives: Advances and perspectives.

D-Galactose derivatives, including galactosyl-conjugates and galactose-upgrading compounds, provide various physiological benefits and find applications in industries such as food, cosmetics, feed, pharmaceuticals. Many research on galactose derivatives focuses on identification, characterization, development, and mechanistic aspects of their physiological function, providing opportunities and challenges for the development of practical approaches for synthesizing galactose derivatives. This study focuses on recent advancements in enzymatic biosynthesis of galactose derivatives. Various strategies including isomerization, epimerization, transgalactosylation, and phosphorylation-dephosphorylation were extensively discussed under the perspectives of thermodynamic feasibility, theoretical yield, cost-effectiveness, and by-product elimination. Specifically, the enzymatic phosphorylation-dephosphorylation cascade is a promising enzymatic synthesis route for galactose derivatives because it can overcome the thermodynamic equilibrium of isomerization and utilize cost-effective raw materials. The study also elucidates the existing challenges and future trends in enzymatic biosynthesis of galactose derivatives. Collectively, this review provides a real-time summary aimed at promoting the practical biosynthesis of galactose derivatives through enzymatic catalysis.

Biocatalytic cascade to polysaccharide amination.

BACKGROUND: Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated. RESULTS: Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure. CONCLUSIONS: Using oxidase-amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme.

Enzymatic cascade reactors on carbon nanotube transistor detecting trace prostate cancer biomarker.

Biosensors based on carbon nanotube field-effect transistors (CNT-FETs) have shown great potential in biomarker detection due to their high sensitivity because of appreciable semiconducting electrical properties. However, background signal interferences in complex mediums may results in low signal-to-noise ratio, which may impose challenges for precise biomarker detection in physiological fluids. In this work, we develop an enzymatic CNT-FET, with scalable production at wafer scale, for detection of trace sarcosine that is a biopsy-correlated biomarker of prostate cancer. Enzymatic cascade rectors are constructed on the CNT to improve the reaction efficiency, thereby, enhancing the signal transduction. As such, a limit of detection as low as 105 zM is achieved in buffer solution. Owing to the enhanced reaction efficiency, the testing of clinical serum samples yields significant signal difference to discriminate the prostate cancer (PCa) samples from the benign prostatic hyperplasia (BPH) samples (P = 1.07 × 10-5), demonstrating immense potential in practical applications.

A circuitous route for in vitro multi-enzyme cascade production of cytidine triphosphate to overcome the thermodynamic bottleneck.

Cytidine triphosphate (CTP), as a substance involved in the metabolism of phospholipids, proteins and nucleic acids, has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline. In this study, we established an in vitro six-enzyme cascade system to generate CTP. To avoid thermodynamic bottlenecks, we employed a circuitous and two-stage reaction strategy. Using cytidine as the key substrate, the final product CTP is obtained via the deamination and uridine phosphorylation pathways, relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate. Several extremophilic microbial-derived deaminases were screened and characterized, and a suitable cytidine deaminase was selected to match the first-stage reaction conditions. In addition, directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition, along with a 1.7-fold increase in activity over the wild type. After optimizing the reaction conditions, we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM, and the yield of CTP exceeded 82% within 10.0 h.

Gas Cluster Ion Beam-Assisted Deposition for Fabricating Dry Multilayers Containing Enzyme and Substrate with On-Demand Release.

Gas cluster ion beam (GCIB)-assisted deposition is used to build multilayered protein-based structures. In this process, Ar3000-5000+ clusters bombard and sputter molecules from a reservoir (target) to a collector, an operation that can be sequentially repeated with multiple targets. The process occurs under a vacuum, making it adequate for further sample conservation in the dry state, since many proteins do not have long-term storage stability in the aqueous state. First of all, the stability in time and versatility in terms of molecule selection are demonstrated with the fabrication of peptide multilayers featuring a clear separation. Then, lysozyme and trypsin are used as protein models to show that the activity remaining on the collector after deposition is linearly proportional to the argon ion dose. The energy per atom (E/n) of the Ar clusters is a parameter that was also changed for lysozyme deposition, and its increase negatively affects activity. The intact detection of larger protein molecules by SDS-PAGE gel electrophoresis and a bioassay (trypsin at ≈25 kDa and glucose oxidase (GOx) at ≈80 kDa) is demonstrated. Finally, GOx and horseradish peroxidase, two proteins involved in the same enzymatic cascade, are successively deposited on ß-d-glucose to build an on-demand release material in which the enzymes and the substrate (ß-d-glucose) are combined in a dry trilayer, and the reaction occurs only upon reintroduction in aqueous medium.

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

BACKGROUND: l-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. RESULTS: Here, 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. SIGNIFICANCE: The 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.

Whole-cell one-pot biosynthesis of dodecanedioic acid from renewable linoleic acid.

BACKGROUND: Dodecanedioic acid (DDA), a typical medium-chain dicarboxylic fatty acid with widespread applications, has a great synthetic value and a huge industrial market demand. Currently, a sustainable, eco-friendly and efficient process is desired for dodecanedioic acid production. RESULTS: Herein, a multi-enzymatic cascade was designed and constructed for the production of DDA from linoleic acid based on the lipoxygenase pathway in plants. The cascade is composed of lipoxygenase, hydroperoxide lyase, aldehyde dehydrogenase, and unidentified double-bond reductase in E. coli for the main cascade reactions, as well as NADH oxidase for cofactor recycling. The four component enzymes involved in the cascade were co-expressed in E. coli, together with the endogenous double-bond reductase of E. coli. After optimizing the reaction conditions of the rate-limiting step, 43.8 g L- 1 d- 1 of DDA was obtained by a whole-cell one-pot process starting from renewable linoleic acid. CONCLUSIONS: Through engineering of the reaction system and co-expressing the component enzymes, a sustainable and eco-friendly DDA biosynthesis route was set up in E. coli, which afforded the highest space time yield for DDA production among the current artificial multi-enzymatic routes derived from the LOX-pathway, and the productivity achieved here ranks the second highest among the current research progress in DDA biosynthesis.

Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning.

The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ1-dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ1-dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (kcat/Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid.

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

BACKGROUND: Organophosphorus 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. RESULTS: Here, 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. SIGNIFICANCE: Furthermore, 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.

In vitro biosynthesis of ATP from adenosine and polyphosphate.

Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0-9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively.

Vía de las lectinas, una ruta del complemento en construcción / Lectin pathway, a complement route under construction

ntroducción: El sistema del complemento puede ser activado por tres vías: clásica, alternativa y de las lectinas, esta última en fase de estudio para su completamiento. Objetivo: Describir hasta donde se ha avanzado en la construcción de la vía de las lectinas, sus iniciadores, activadores, reguladores, cascada enzimática y sus funciones biológicas. Metodología: Se realizó una revisión sobre el tema en estudio empleando artículos de libre acceso en la base de datos Pubmed y los trabajos publicados por el grupo de trabajo de la Universidad de Goettigen, la Universidad de Aarhus en Dinamarca y el Laboratorio Central de Líquido Cefalorraquídeo (LABCEL) de la Universidad de Ciencias Médicas de La Habana en los últimos cinco años comprendidos en el período de enero de 2012 a marzo del 2017. Desarrollo: Los iniciadores de la vía de las lectinas son las moléculas de reconocimiento colectinas y ficolinas circulantes en sangre, que participan en muchos procesos del organismo. Los activadores de esta vía son las MASP 1, 2 presentes como proenzimas; y la MASP 3, MAp 19 y 44 actúan como reguladoras. La cascada enzimática luego del reconocimiento es similar a la ruta clásica. Conclusiones: Las colectinas y ficolinas inician la vía de las lectinas. Sus activadores son las MASP 1, 2. Los reguladores son la MASP-3, y las MAp 19 y 44. Similar a la clásica en su cascada enzimática. Es la más antigua en la filogenia por eso participa en muchos procesos en el organismo(AU)

Introduction. The complement system can be activated in three ways: classical, alternative and lectins, the latter in the study phase for its completion. Objective. To describe the progress made in the construction of the lectin pathway, its initiators, activators, regulators, enzymatic cascade and its biological functions. Methods. A review was made on the subject under study using articles of free access in the Pubmed database and the works published by the working group of the University of Goettigen, the University of Aarhus in Denmark and the Central Laboratory of Cefalorraquìdeo liquid (LABCEL) of the University of Medical Sciences of Havana in the last five years included in the period from January 2012 to March 2017. Development. The initiators of the lectin pathway are the collectin recognition molecules and circulating ficolins in blood, which participate in many processes of the organism. The activators of this pathway are MASP 1, 2 present as proenzymes; and MASP 3, MAp 19 and 44 act as regulators. The enzymatic cascade after recognition is similar to the classical route. Conclusions. Collectins and ficolines initiate the lectin pathway. Its activators are MASP 1, 2. The regulators are MASP-3, and MAp 19 and 44. Similar to the classic in its enzymatic cascade. It is the oldest in phylogeny so it participates in many processes in the body.(AU)