Enzymatic Process Research Articles

Cross-Sectional Analysis of Hypoxia-Regulated miRNA-181a, miRNA-199a, HIF-1α, and SIRT1 in the Development of Type 2 Diabetes in Patients with Obstructive Sleep Apnea—Preliminary Study

Introduction: Obstructive sleep apnea (OSA) is recognized as an independent risk factor for diabetes mellitus type 2 (T2DM) development, which is twice as common in patients with OSA compared to non-OSA patients. Objectives: This study aimed to investigate changes in oxygen metabolism and their role in T2DM development among OSA patients through epigenetic processes via miRNA-181a, miRNA-199a, and enzymatic processes via SIRT1 and HIF-1α. Methods: Based on polysomnography, apnea–hypopnea index and the presence of T2DM patients were divided into three groups: control group (n = 17), OSA group (n = 11), OSA&T2DM (n = 20) group. Total RNA was extracted from the buffy coat. Moreover, HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) was counted. Results: Morning miRNA-181a expression was significantly higher in the OSA&T2DM group than in the control group: 67.618 vs. 32.685 (p = 0.036). Evening miRNA-199a expression was significantly higher in the OSA group than in the control group: 5.043 vs. 2.081 (p = 0.042), while its morning expression was significantly higher in the OSA&T2DM group when compared to the control: 4.065 vs. 1.605 (p = 0.036). MiRNA-181a evening expression revealed a negative correlation with the SIRT1 evening and morning expressions (R = −0.367, p = 0.010 and R = −0.405, p = 0.004, respectively). Moreover, morning miRNA-181a was positively correlated with HOMA-IR (R = 0.321, p = 0.034). MiRNA-199a evening expression presented a moderate positive correlation with the SIRT1 morning expressions (R = 0.48, p < 0.001) and HOMA-IR (R = 0.35, p = 0.02). Conclusions: Patients suffering from OSA and T2DM had an increased expression of miRNA-181a. Moreover, a negative correlation between miRNA-181a and SIRT1 expression was observed, while a correlation between miRNA-181a and insulin resistance was positive. This phenomenon might suggest a possible epigenetic pathway for an increased incidence of T2DM in OSA patients however further research is needed.

Molecular Decoration and Unconventional Double Bond Migration in Irumamycin Biosynthesis

Irumamycin (Iru) is a complex polyketide with pronounced antifungal activity produced by a type I polyketide (PKS) synthase. Iru features a unique hemiketal ring and an epoxide group, making its biosynthesis and the structural diversity of related compounds particularly intriguing. In this study, we performed a detailed analysis of the iru biosynthetic gene cluster (BGC) to uncover the mechanisms underlying Iru formation. We examined the iru PKS, including the domain architecture of individual modules and the overall spatial structure of the PKS, and uncovered discrepancies in substrate specificity and iterative chain elongation. Two potential pathways for the formation of the hemiketal ring, involving either an olefin shift or electrocyclization, were proposed and assessed using 18O-labeling experiments and reaction activation energy calculations. Based on our findings, the hemiketal ring is likely formed by PKS-assisted double bond migration and TE domain-mediated cyclization. Furthermore, putative tailoring enzymes mediating epoxide formation specific to Iru were identified. The revealed Iru biosynthetic machinery provides insight into the complex enzymatic processes involved in Iru production, including macrocycle sculpting and decoration. These mechanistic details open new avenues for a targeted architecture of novel macrolide analogs through synthetic biology and biosynthetic engineering.

Discovery of Non-Covalent Inhibitors for SARS-CoV-2 PLpro: Integrating Virtual Screening, Synthesis, and Experimental Validation.

The SARS-CoV-2 pandemic has significantly challenged global public health, highlighting the need for effective therapeutic options. This study focuses on the papain-like protease (PLpro) of SARS-CoV-2, which is a critical enzyme for viral polyprotein processing, maturation, and immune evasion. We employed a combined approach that began with computational models in a virtual screening campaign, prioritizing compounds from our in-house chemical library against PLpro. Out of 81 virtual hits evaluated through enzymatic and biophysical assays, we identified a modest inhibitor featuring a naphthyridine core with an IC50 of 73.61 μM and a K i of 22 μM. Expanding our exploration, we synthesized and assessed 30 naphthyridine analogues, three of which emerged as promising noncovalent, nonpeptidomimetic inhibitors with IC50 values between 15.06 and 51.81 μM. Furthermore, in vitro ADMET assays revealed these compounds to possess moderate aqueous solubility, low cytotoxicity, and high microsomal stability, making them excellent candidates for further development targeting SARS-CoV-2 PLpro.

A novel mechanocatalytic process by vibratory disk mill for efficient hydrolysis of cellulose, chitin and xylan

Polysaccharides are primary sources to manufacture chemical products with low carbon footprints, owing to their carbons from atmospheric carbon dioxide fixed through photosynthesis. Breaking down such polysaccharides into water-soluble saccharides through the hydrolysis of glycosidic bonds is one of key steps to produce valuable chemicals; however, previous chemical and enzymatic processes have been suffered from their fundamental drawbacks (e.g. not environmentally benign, high production cost) and are not regarded as feasible approaches. Recently, mechanocatalytic process, which proceeds hydrolysis of polysaccharides by simply milling with solid acids, attracts attentions owing to its cost-effectiveness and environmental beingness. Herein, we propose the novel mechanocatalytic system to efficiently proceed the hydrolysis of glycosidic bonds in polysaccharides by using “vibratory disk mill”, which can produce strong shear and impact force. By milling polysaccharides (cellulose, chitin and xylan) with solid acid kaolin, the polysaccharides can be efficiently hydrolysed to water-soluble saccharides with a rate twice higher than the previous milling devices, achieving quantitative conversion of polysaccharide to water soluble products (solubility: >99 %). We elucidated the reaction mechanism of polysaccharides with kaolin by X-ray diffractograms: forces produced by the vibratory disk mill can efficiently delaminate layered aluminosilicate nanosheets in kaolin, leading to better accessibility of polysaccharides on the acidic surface of the nanosheets. This novel mechanocatalytic system can also be applicable to produce oligosaccharides directly from crude biomass samples (birchwood chips and shrimp shells) without any chemical purification steps, demonstrating that this system can be used in industrial biomass processing.

Molecular dynamics studies reveal the structural impacts of LRRK2 R1441C and LRRK2 D1994A mutations in Parkinson's disease

Parkinson's Disease (PD) is a continuingly deteriorating neurological ailment affecting over 8.5 million patients globally as of 2019, and the numbers are expected to keep rising. To aid in identifying therapeutic targets, molecular dynamics simulations are convenient and cost-effective methods for enriching our knowledge of the molecular pathophysiology of diseases. Many proteins and their corresponding mutations have been identified to contribute to this disease, of which Leucine-rich repeat kinase 2 (LRRK2) is accountable for a significant percentage. Several mutations involving the domains in LRRK2 have been studied, which are known to interfere with various enzymatic processes, ultimately leading to trademark features of PD like aggregation of protein inclusions called Lewy Bodies (LBs), mitochondrial dysfunctions, etc. The precise molecular mechanism of the mutations' pathophysiology is still unclear. This research article looks at the structural effects of mutations, namely the R1441C and D1994A mutations, on the surrounding residues in the protein, offering novel insights into pathophysiological changes at an atomistic level. Our results indicate a gain of electrostatic interactions with a stable αβ motif within the LRR-Roc linker, amongst other changes. This article also highlights the potential involvement and importance of the αβ motif in LRRK2 associated PD.

Role of Enzymes in Food Processing

Enzymes are very important in the food industry because they naturally speed up and improve various processes. They are used in many areas, including baking, dairy, brewing, and the processing of fruits and vegetables. Enzymes help make food products better in quality, longer-lasting, more stable, and tastier. In baking, enzymes like amylases, proteases, and lipases are essential. Amylases turn starches into sugars, which feed the yeast and help the dough rise more effectively. Proteases change gluten, making the dough easier to work with. Lipases improve the texture and softness of the bread, helping it stay fresh for a longer time. In dairy processing, enzymes such as rennet and lactase are crucial. Rennet is a mix of enzymes used in cheese-making to coagulate milk, which leads to the creation of curds and whey. Lactase breaks down lactose into glucose and galactose, making dairy products suitable for people who are lactose intolerant. These enzymatic actions not only improve the nutritional value of dairy products but also enhance their digestibility and flavouring, in the brewing industry, enzymes like amylases, proteases, and glucanases are used to convert starches into fermented sugars, break down proteins for better clarity, and lower viscosity. These enzymes help with efficient fermentation, resulting in higher alcohol yields and better beer quality. Additionally, β-glucanase helps break down β-glucans, which can cause problems during filtration, thus improving the overall brewing process. When it comes to processing fruits and vegetables, enzymes such as pectinases, cellulases, and hemicelluloses play important roles. Pectinases break down pectin, a key component of plant cell walls, leading to clearer juices and more efficient juice extraction. Cellulases and hemicelluloses further assist by breaking down cell walls, which helps release valuable nutrients and improves the texture of the processed products. Enzymes are essential in the food processing industry, providing numerous benefits such as better product quality, longer shelf life, increased nutritional value, and greater efficiency in production. Their ability to work under mild conditions, along with their specificity and biodegradability, makes them ideal for sustainable food production. Continuous research and the development of new enzymes and their applications will keep transforming the industry, leading to healthier, safer, and more sustainable food products.

A Fluorescent Perspective on Water Structuring: ACDAN in Salt Solutions and Hydrogels

The interactions and structural organization of water molecules play a crucial role in a wide range of physical, chemical, and biological processes. The ability of water to form hydrogen bonds (H-bonds) underpins its unique properties and enables it to respond dynamically to various environmental factors. These interactions at the molecular level may affect vital processes like protein folding, enzyme activity, and cellular organization. The presence of solutes and spatial constraints can alter the H-bonding network of water, and these effects are ubiquitous in the biological environment. In this study, we analyzed the fluorescence of 2-acetyl-6-(dimethylamino)naphthalene (ACDAN) fluorescence emission in water solutions containing kosmotropic and chaotropic salts and in agar hydrogels. Recently, this dye has proven invaluable in studying water network structure and dynamics, as its fluorescence signal changes based on the local dielectric environment, revealing variations in the dipolar relaxation of water. Our results show that ACDAN spectral response correlates with the degree of water ordering, providing important insights into solute–water interactions and water dynamics in free and confined environments.

Loss of peroxiredoxin 6 (PRDX6) alters lipid composition and distribution resulting in increased sensitivity to ferroptosis.

Peroxiredoxin 6 (PRDX6) is a multifunctional enzyme involved in phospholipid peroxide repair and metabolism. In this study we investigated the global lipid composition of a human hepatocarcinoma cell line SNU475 lacking PRDX6 and lipid related cellular processes. There was a general decrease in multiple lipids species upon loss of PRDX6, in particular sphingomyelins and acylcarnitines, consistent with previously observed alterations in cell signaling pathways and mitochondrial dysfunction. Deprivation of docosahexaenoic acid and related species was also evident. However, a few striking exceptions are worth highlighting: 1) Three specific arachidonic acid (AA) containing phophatidylcholines (PC) increased significantly. The increase of sn1-stearic/sn2-PUFA containing PC and sn2-AA containing plasmenyls are indicative of a preference of PRDX6 iPLA2 activity for these AA storage glycerophospholipids. 2) Several polyunsaturated fatty acids (PUFA) and PUFA containing triacylglycerols accumulated together with increased formation of lipid droplets, an indication of altered FA flux and PUFA sequestration in PRDX6 knockout cells. Loss of PRDX6 resulted in increased sensitivity to erastin-induced ferroptosis, independent of selenium and GPX4, as a consequence of increased levels of lipid hydroperoxides, that reverted to normal levels upon rescue with PRDX6. <SPAN style="font-weight: 400;">The results presented demonstrate that all three enzymatic activities of PRDX6 contribute to the role of this multifunctional enzyme in diverse cellular processes, including membrane phospholipid remodeling and glycerophospholipid functional diversity, resulting in altered lipid peroxides and modulation of AA disposition and traffic. These contributions highlight the complexity of the changes that loss of PRDX6 exerts on cell functionality.</SPAN>.

Precision Activity-Based α-Amylase Probes for Dissection and Annotation of Linear and Branched-Chain Starch-Degrading Enzymes.

α-Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti-diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α-amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α-amylase function. Here, we design, synthesize and apply new branched α-amylase activity-based probes. Using both α-1,6 branched and unbranched α-1,4 maltobiose activity-based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α-amylases and a panel of industrially relevant α-amylases. We also demonstrate how we can detect and annotate the substrate specificity of α-amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in-gel fluorescence and mass spectrometry. A toolbox of starch-active activity-based probes will enable rapid functional dissection of α-amylases. We envisage activity-based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health.

Enzymatic conversion of wood materials from the pulp and paper industry

The reactivity during enzymatic hydrolysis of 8 industrially produced samples of pulps and semi-chemical pulps by enzyme preparations of glycosyl hydrolases B151 and F10 produced by a strain of ascomycete fungus Penicillium verruculosum has been determined. It is shown for the first time that among fibrous pulps available on the market of pulp and paper industry in Russia, the highest level of yield of glucose from the initial wood during biocatalysis using cellulases and hemicellulases is characteristic of semi-chemical pulps obtained after cooking of hardwood with green liquor. A high degree of enzymatic conversion of softwood bleached kraft pulp has been established, which in combination with the possibility of obtaining modified polysaccharide materials from non-hydrolysable residue makes this cellulosic substrate the most promising for the development of biological processes at pulp and paper industries. It is shown that drying of pulp negatively affects the efficiency of cellulose hydrolysis, while mechanical milling improves the performance of the enzymatic saccharification process.

Phosphorus dynamics and sustainable agriculture: The role of microbial solubilization and innovations in nutrient management

Phosphorus (P) is an essential element for plant growth, playing a crucial role in various metabolic processes. Despite its importance, phosphorus availability in soils is often restricted due to its tendency to form insoluble complexes, limiting plant uptake. The increasing demand for phosphorus in agriculture, combined with limited global reserves of phosphate rock, has created challenges for sustainable plant production. Additionally, the overuse of chemical phosphorus fertilizers has resulted in environmental degradation, such as eutrophication of water bodies. Increasing agronomic phosphorus (P) efficiency is crucial because of population growth and increased food demand. Hence, microorganisms involved in the P cycle are a promising biotechnological strategy that has gained global interest in recent decades. Microorganisms' solubilization of phosphate rock (PR) is an environmentally sustainable alternative to chemical processing for producing phosphate fertilizers. Phosphorus-solubilizing microorganisms (PSMs), including bacteria and fungi, and their enzymatic processes offer an eco-friendly and sustainable alternative to chemical inputs by converting insoluble phosphorus into forms readily available for plant uptake. Integrating PSMs into agricultural systems presents a promising strategy to reduce dependence on chemical fertilizers, enhance soil health, and contribute to the transition toward more sustainable and resilient agricultural practices. It can be an alternative that reduces the loss of phosphorus in the environment, especially the eutrophication of aquatic systems. This paper explores the challenges of phosphorus availability in agriculture and the potential of microbial phosphorus solubilization as a sustainable alternative to conventional practices.

Correlating enzymatic reactivity for different substrates using transferable data-driven collective variables

Machine learning (ML) is transforming the investigation of complex biological processes. In enzymatic catalysis, one significant challenge is identifying the reactive conformations (RC) of the enzyme:substrate complex where the substrate assumes a precise arrangement in the active site necessary to initiate a reaction. Traditional methods are hindered by the complexity of the multidimensional free energy landscape involved in the transition from nonreactive to reactive conformations. Here, we applied ML techniques to address this challenge, focusing on human pancreatic α-amylase, a crucial enzyme in type-II diabetes treatment. Using ML-based collective variables (CVs), we correlated the probability of being in a RC with the experimental catalytic activity of several malto-oligosaccharide substrates. Our findings demonstrate a remarkable transferability of these CVs across various compounds, significantly streamlining the modeling process and reducing both computational demand and manual intervention in setting up simulations for new substrates. This approach not only advances our understanding of enzymatic processes but also holds substantial potential for accelerating drug discovery by enabling rapid and accurate evaluation of drug efficacy across different generations of inhibitors.

Advances and Challenges in the Development of Immobilized Enzymes for Batch and Flow Biocatalyzed Processes.

The development of immobilized enzymes both for batch and continuous flow biocatalytic processes has gained significant traction in recent years, driven by the need for cost-effective and sustainable production methods in the fine chemicals and pharmaceutical industries. Enzyme immobilization not only enables the recycling of biocatalysts but also streamlines downstream processing, significantly reducing the cost and environmental impact of biotransformations. This review explores recent advancements in enzyme immobilization techniques, covering both carrier-free methods, entrapment strategies and support-based approaches. At this regard, the selection of suitable materials for enzyme immobilization is examined, highlighting the advantages and challenges associated with inorganic, natural, and synthetic organic carriers. Novel opportunities coming from innovative binding strategies, such as genetic fusion technologies, for the preparation of heterogeneous biocatalysts with enhanced activity and stability will be discussed as well. This review underscores the need for ongoing research to address current limitations and optimize immobilization strategies for industrial applications.

From Batch to Continuous Flow Synthesis in Enzymatic Process Towards Molnupiravir.

Molnupiravir (1) is one among the limited therapeutic options for treating COVID-19 infection and exhibits pan-antiviral potency. Because of urgent demands during the COVID-19 pandemic, a number of methods were developed to offer more efficient routes. In this report, we present a facile 2-step and scalable synthesis of molnupiravir for batch processing and show the implementation of continuous flow biocatalysis to improve the efficiency in synthesis. Our key step entails immobilized lipase and isobutyric anhydride to facilitate regioselective esterification. In batch process, transamination of cytidine (2) provides N4-hydroxycytidine (NHC, 3) with 75 % yield followed by esterification of NHC to give molnupiravir with 64 % yield, providing 48 % overall yield and 99.98 % purity (HPLC). Compared to batch approach in the esterification step, the continuous flow process provides similar product yield and purity and highlights the advantages including 2.42-fold better productivity (mol/day), 2.47-fold improved reaction time, and 30-fold higher space-time-yield. The optimized batch and continuous flow biocatalysis enhance synthesis efficiency and reduce environmental impact, offering a sustainable approach for industrial molnupiravir production.

Application of High-pressure Carbon Dioxide in Japanese Anchovy Waste Recycling by Enzymatic Hydrolysis.

Anchovy waste, a protein resource with high nutritional value and potential for recycling with a relatively high economic effect, is essential for the Sustainable Development Goals of the United Nations. Preventing microbial contamination during the recycling process, through enzymatic hydrolysis, ensures the safety of recycled products. High-pressure carbon dioxide is a novel non-thermal decontamination technology, which inactivates cells by breaking their membranes. Here, we selected 40 °C_5.0 MPa and 50 °C_1.0 MPa treatment conditions for effectively decontaminating anchovy samples during the hydrolysis process. Next Generation Sequencing and real-time PCR experiments showed that a microbial growth promotion stage existed at the beginning of 40 °C_5.0 MPa, which may threaten hydrolysates, as some microbial genera were detected from the metabolites produced. Treatment at 50 °C_1.0 MPa ensured a high safety level for hydrolysates but this is limiting for various enzymatic hydrolysis processes. Orientaaze OP was selected as an additional enzyme with the highest hydrolysis efficiency under 40 and 50 °C among 10 different industrial proteases. Compared with control samples without high-pressure carbon dioxide treatment, 40 °C_5.0 MPa and 50 °C_1.0 MPa treated samples presented higher total amino acid concentrations by ultra-high-performance liquid chromatography. Hence, there was an increased enzyme activity by 40 °C_5.0 MPa and 50 °C_1.0 MPa treatments in endogenous or additional proteases hydrolytic processes. Despite the need for more future studies to be conducted, this research still provides essential information and instruction for industrial enzymatic hydrolysis applications on anchovy waste recycling.

Effects of different straw return methods on soil properties and yield potential of maize

Long-term continual straw return can enhance soil quality and increase crop yields by perpetually altering the soil environment. However, little is known about how different straw return methods affect soil physicochemical properties, enzymatic processes, and crop yields. The study aims to determine how different straw return practices improve soil structure, nutrients, enzyme activities, and maize yields. In this experiment, a field trial was conducted in 2021–2022 in the irrigated area of the Tumochuan Plain Irrigation District to determine the effects of four different straw returns on soil structure, nutrients, enzyme activities, soil quality, and maize yields. The four types of straw return included straw incorporation with deep tillage (DPR), straw incorporation with subsoiling (SSR), no-tillage mulching straw return, and farmer’s shallow rotation (CK). Our results showed that DPR and SSR enhanced water retention capacity by reducing the bulk weight of the 0–45 cm soil layer. DPR and SSR significantly increased soil organic C (12.76%), total nitrogen (25.32%), and available nutrients (i.e. AP, NO3−–N) in the 0–45 cm soil layer compared to CK, whereas there were no differences between straw-returned treatments in the 0–15 cm soil layer. Finally, maize yield was significantly increased by 13.14% and 11.41% in the second year of DPR and SSR, respectively, compared to CK. This study demonstrated that DPR and SSR are effective at enhancing the agricultural utilization of crop residues and represent feasible strategies for improving physical, chemical, and biological processes in continuous maize cropping systems, leading to increased crop productivity.

Fermentation for Revalorisation of Fruit and Vegetable By-Products: A Sustainable Approach Towards Minimising Food Loss and Waste.

In a world increasingly focused on sustainability and integrated resource use, the revalorisation of horticultural by-products is emerging as a key strategy to minimise food loss and waste while maximising value within the food supply chain. Fermentation, one of the earliest and most versatile food processing techniques, utilises microorganisms or enzymes to induce desirable biochemical transformations that enhance the nutritional value, digestibility, safety, and sensory properties of food products. This process has been identified as a promising method for producing novel, high-value food products from discarded or non-aesthetic fruits and vegetables that fail to meet commercial standards due to aesthetic factors such as size or appearance. Besides waste reduction, fermentation enables the production of functional beverages and foods enriched with probiotics, antioxidants, and other bioactive compounds, depending on the specific horticultural matrix and the types of microorganisms employed. This review explores the current bioprocesses used or under investigation, such as alcoholic, lactic, and acetic acid fermentation, for the revalorisation of fruit and vegetable by-products, with particular emphasis on how fermentation can transform these by-products into valuable foods and ingredients for human consumption, contributing to a more sustainable and circular food system.

Allopolyploidy expanded gene content but not pangenomic variation in the hexaploid oilseed Camelina sativa.

Ancient whole-genome duplications (WGDs) are believed to facilitate novelty and adaptation by providing the raw fuel for new genes. However, it is unclear how recent WGDs may contribute to evolvability within recent polyploids. Hybridization accompanying some WGDs may combine divergent gene content among diploid species. Some theory and evidence suggest that polyploids have a greater accumulation and tolerance of gene presence-absence and genomic structural variation, but it is unclear to what extent either is true. To test how recent polyploidy may influence pangenomic variation, we sequenced, assembled, and annotated twelve complete, chromosome-scale genomes of Camelina sativa, an allohexaploid biofuel crop with three distinct subgenomes. Using pangenomic comparative analyses, we characterized gene presence-absence and genomic structural variation both within and between the subgenomes. We found over 75% of ortholog gene clusters are core in Camelina sativa and <10% of sequence space was affected by genomic structural rearrangements. In contrast, 19% of gene clusters were unique to one subgenome, and the majority of these were Camelina-specific (no ortholog in Arabidopsis). We identified an inversion that may contribute to vernalization requirements in winter-type Camelina, and an enrichment of Camelina-specific genes with enzymatic processes related to seed oil quality and Camelina's unique glucosinolate profile. Genes related to these traits exhibited little presence-absence variation. Our results reveal minimal pangenomic variation in this species, and instead show how hybridization accompanied by WGD may benefit polyploids by merging diverged gene content of different species.

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Simultaneous One-Step Preparation of β/κ-Carrapentaose and 3,6-Anhydro-D-galactose by Cascading κ-Carrageenase and an Exo-α-3,6-Anhydro-D-galactosidase.

Carrageenan oligosaccharides have shown promising bioavailability and possess a variety of physiological activities, making them highly suitable for use in the food, pharmaceutical, and agricultural industries. The preferred method for producing carrageenan oligosaccharides is using various carrageenolytic enzymes, as it offers mild reaction conditions, high efficiency, and product specificity. However, there is still a lack of specific applications for using these enzymes to prepare odd-numbered carrageenan-oligosaccharides (OCOSs). Our previous research identified a more convenient route for simultaneously preparing OCOSs and 3,6-anhydro-D-galactose (D-AHG) using only two types of carrageenolytic enzymes: κ-carrageenase and exo-α-3,6-anhydro-D-galactosidase (D-ADAGase). In this study, we utilized a CipA-based self-assembly system to cascade κ-carrageenase CaKC16A and D-ADAGase ZuGH129A for one-step preparation of β/κ-carrapentaose, G-(DA-G4S)2, and D-AHG from degrading β/κ-carrageenan. This self-assembled enzyme, namely CipA-CaKC16A-ZuGH129A, can be easily obtained through a simple centrifugation process. The final optimized enzymatic process produced 0.74 g/L G-(DA-G4S)2 and 0.13 g/L D-AHG. This cascade system of different types of carrageenolytic enzymes has the potential to achieve the preparation of various types of carrageenan oligosaccharides.