Redox Enzymes Research Articles

Microbial consortia driving (ligno)cellulose transformation in agricultural woodchip bioreactors

ABSTRACT Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors and their active enzymes. Our findings highlight a microbial community enriched for (ligno)cellulose-degrading denitrifiers with key players from the taxa Giesbergeria , Cellulomonas , Azonexus, and UBA5070 ( Fibrobacterota ). A wide substrate specificity is observed among the many expressed carbohydrate-active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions. IMPORTANCE Freshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria , Cellulomonas , Azonexus , and UBA5070 ( Fibrobacterota ) playing significant roles in lignocellulose transformation and showcasing a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improve water quality, protect aquatic ecosystems, and reduce greenhouse gas emissions from WBRs.

Controlled Stimulus-Responsive Delivery Systems for Osteoarthritis Treatment

Osteoarthritis (OA), a common and disabling degenerative joint disease, affects millions of people worldwide and imposes a considerable burden on patients and society due to its high prevalence and economic costs. The pathogenesis of OA is closely related to the progressive degradation of articular cartilage and the accompany inflammation; however, articular cartilage itself cannot heal and modulate the inflammation due to the lack of nerves, blood vessels, and lymph-vessels. Therefore, reliable and effective methods to treat OA remain highly desired. Local administration of drugs or bioactive materials by intra-articular injection of the delivery system represents a promising approach to treat OA, especially considering the prolonged joint retention, cartilage or chondrocytes targeting, and stimuli-responsive release to achieve precision OA therapy. This article summarizes and discusses the advances in the currently used delivery systems (nanoparticle, hydrogel, liposome, and microsphere) and then focuses on their applications in OA treatment from the perspective of endogenous stimulus (redox reactions, pH, enzymes, and temperature) and exogenous stimulus (near-infrared, magnetic, and ultrasound)-responsive release. Finally, the challenges and potential future directions for the development of nano-delivery systems are summarized.

Tethered heme domains in a triheme cytochrome allow for increased electron transport distances.

Decades of research describe myriad redox enzymes that contain cofactors arranged in tightly packed chains facilitating rapid and controlled intra-protein electron transfer. Many such enzymes participate in extracellular electron transfer (EET), a process which allows microorganisms to conserve energy in anoxic environments by exploiting mineral oxides and other extracellular substrates as terminal electron acceptors. In this work, we describe the properties of the triheme cytochrome PgcA from Geobacter sulfurreducens. PgcA has been shown to play an important role in EET but is unusual in containing three CXXCH heme binding motifs that are separated by repeated (PT)x motifs, suggested to enhance binding to mineral surfaces. Using a combination of structural, electrochemical, and biophysical techniques, we experimentally demonstrate that PgcA adopts numerous conformations stretching as far as 180 Å between the ends of domains I and III, without a tightly packed cofactor chain. Furthermore, we demonstrate a distinct role for its domain III as a mineral reductase that is recharged by domains I and II. These findings show PgcA to be the first of a new class of electron transfer proteins, with redox centers separated by some nanometers but tethered together by flexible linkers, facilitating electron transfer through a tethered diffusion mechanism rather than a fixed, closely packed electron transfer chain.

Hybrid Type I and II Polyketide Synthases Yield Distinct Aromatic Polyketides.

Bacterial aromatic polyketides are compounds with multiple aromatic rings synthesized by bacterial type II polyketide synthases (PKSs), some of which have been developed into clinical drugs. Compounds containing aromatic polyketides synthesized by hybrid type I and type II PKSs are extremely rare. Here, we report the discovery of a gene cluster encoding both modular type I and type II PKSs as well as KAS III through extensive bioinformatics analysis, leading to the characterization of the hybrid polyketide, spirocycline A. The structure of spirocycline A is rare among all aromatic polyketides, featuring a unique starter unit and four spirocycles and forming a dimer. Biosynthetic studies indicate that the starter unit of this molecule is synthesized by type I PKS in collaboration with two trans-acting ketoreductase (KR) and enoylreductase (ER). It is then transferred by KAS III to the type II PKS system, which synthesizes the tricyclic aromatic polyketide backbone. The subsequent formation of the spirocycle and dimerization are carried out by four redox enzymes encoded in the gene cluster. Overall, the discovery of spirocycline A provides a new approach for identifying novel aromatic polyketides and offers potential enzymatic tools for the bioengineering of these hybrid polyketides.

Current summer heat waves impair rainbow trout (Oncorhynchus mykiss) spermatogenesis: Implications for future fish farming management practices in South Europe

Freshwater environments provide different ecosystem services for human well-being, and are one of the most important natural sources of healthy animal protein. Climate change and extreme temperature events, among other factors, represent a growing concern for their sustainable use. In contrast to the research effort to elucidate how global warming affects these aquatic environments, few studies pay attention to the potential effects of heat waves, a current phenomenon with direct effects on contemporaneous organisms. Here, the potential effects of a single heat wave event on rainbow trout (Oncorhynchus mykiss) males, mimicking a recorded environmental event, have been evaluated by means of biometric, redox and stress response, plasma testosterone, histopathology and transcriptomic analyses. Environmental monitoring showed how, during the last 4 years, mean water temperature increased (2–4 °C; from April to October), heat wave duration progressively increased (from 15 to 100 days), and mean intensities ranged 1.7–3.8 °C with a maximum of 8.0 °C. An experimentally recreated heat wave lasting only 16 days, with mean and maximum intensities of 2.7 and 6.7 °C, induced a decrease in body weight and total antioxidant status, but increased blood plasma cortisol levels and redox enzyme activity. The induced heat wave decreased testosterone plasma levels and sperm quality, arrested spermatocyte type I and II differentiation and increased spermatozoa apoptosis. These effects were correlated with a differential expression of 1156 genes (116 up- and 1040 down-regulated genes) in exposed fish when compared to Control fish, reflecting an alteration in cell apoptosis and spermatozoa maturation processes. These results not only increase concern about current heat wave events, recreating a real scenario, but also unveil for the first time the particular molecular mechanisms of subsequent spermatogenesis disruption, opening new avenues for designing urgent and necessary strategies to mitigate the effects of extreme climate change conditions on freshwater aquaculture.

Oral toxicity assessment and the mitigation of lung carcinogenesis by phytol and α-bisabolol combination treatment in swiss albino mice: insights into redox enzyme modulation and caspase-dependent cell death mechanisms.

This study examined the safety and potential anti-lung cancer effects of combinations of phytol and α-bisabolol in Swiss albino mice. Both acute and subacute toxicity assessments showed that the combination of phytol and α-bisabolol is safe, with no adverse effects observed at higher concentrations. Hematological, biochemical, and histopathological tests showed no signs of toxicity in the heart, lungs, liver, spleen, and kidneys. The LD50 was greater than 2000mg/kg, indicating a large safety margin. Histopathological analysis confirmed cancer induction in the B(a)P-induced group, which had significantly altered relative lung weights. Lung weight increased slightly pre and post-treatment, but histopathology showed normal alveolar epithelium. GSH and SOD levels increased significantly in B(a)P-exposed groups, indicating an adaptive antioxidant response. CAT levels increased significantly in the post-treatment group, demonstrating the role of combination of phytol and α-bisabolol in protecting against B(a)P-induced oxidative damage. Upregulation of Bax and downregulation of Bcl-2 caused a pro-apoptotic environment, suggesting a way to inhibit malignant cell survival. Modulation of caspase-3 and caspase-9 showed the complexity of carcinogen-induced apoptotic signaling. In conclusion, phytol and α-bisabolol were found to be safe and organ-protective, and demonstrated no acute or subacute toxicity. They modulate antioxidant defenses and apoptotic pathways, which may help prevent and treat lung cancer.

Metabolic Rate and Oxidative Stress as a Risk Factors in the Development of Colorectal Cancer.

There is growing evidence that the body's energy expenditures constitute a significant risk factor for the development of most deadly diseases, including cancer. Our aim was to investigate the impact of basal metabolic rate (BMR) on the growth and progression of colorectal cancer (CRC). To do so, we used a unique model consisting of three lines of laboratory mice (Mus musculus) artificially selected for high (HBMR) and low (LBMR) basal metabolic rate and randomly bred individuals (non-selected, NSBMR). The experimental individuals were implanted with human colorectal cancer cells DLD-1. The variation in BMR between the lines allowed for testing the impact of whole-body metabolism on oxidative and antioxidant parameters in the liver throughout the cancerogenesis process. We investigated the dependence between metabolic values, reactive oxygen species (ROS) levels, and Kelch-like ECH-associated protein 1-based E3 ligase complexes (Keap1) gene activity in these animals. We found that the HBMR strain had a higher concentration of oxidative enzymes compared to the LBMR and NSBMR. Furthermore, the growth rate of CRC tumors was associated with alterations in the levels of oxidative stress enzymes and Keap1 expression in animals with a high metabolic rate. Our results indicate that a faster growth and development of CRC line DLD-1 is associated with enzymatic redox imbalance in animals with a high BMR.

Fully Integrated Biosensing System for Dynamic Monitoring of Sweat Glucose and Real-Time pH Adjustment Based on 3D Graphene MXene Aerogel.

The development of noninvasive glucose sensors capable of continuous monitoring without restricting user mobility is crucial, particularly for managing diabetes, which demands consistent and long-term observation. Traditional sensors often face challenges with accuracy and stability that curtail their practical applications. To address these issues, we have innovatively applied a three-dimensional porous aerogel composed of Ti3C2Tx MXene and reduced graphene oxide (MX-rGO) in electrochemical sensing. It significantly reduces the electron-transfer distance between the enzyme's redox center and the electrode surface while firmly anchoring the enzyme layer to effectively prevent any leakage. Another pivotal advancement in our study is the integration of the sensor with a real-time adaptive calibration mechanism tailored specifically for analyzing sweat glucose. This sensor not only measures glucose levels but also dynamically monitors and adjusts to pH fluctuations in sweat. Such capabilities ensure the precise delivery of physiological data during physical activities, providing strong support for personalized health management.

RECENT ADVANCEMENT IN ELECTROCHEMICAL BIOSENSOR BASED ON REDOX ENZYME COMPOSITE MODIFIED ELECTRODE FOR THE DETECTION OF BIOMOLECULES

Recent developments in electrochemical biosensors have resulted in new applications in a variety of disciplines, such as clinical diagnosis, food processing quality control, and environmental monitoring. The electrochemical biosensor is a widely used sensing device that transmits an electrical signal from biological activities. Electrochemical biosensors have led to significant advancements in detecting various biomolecules such as uric acid, glucose, cholesterol, lactate, and DNA, including cancer biomarkers, viruses, and antibodies. An electrode is a vital part of this kind of sensor since it serves as a strong support for immobilizing biomolecules (enzyme, antibody, and nucleic acid) and allowing electron flow. Redox enzymes are the foundation of enzymatic electrochemical biosensors, which use oxidation or reduction reactions to detect a substrate, which is then converted into an electrical signal. Immobilizing enzymes onto solid supports is a common method to enhance the performance of an enzymatic electrochemical biosensor. However, there are some challenges, such as potential loss of enzyme activity, difficulties in maintaining enzyme stability, and issues related to the reproducibility and consistency of enzyme immobilization. This review covers all challenges in enzymatic electrochemical biosensors. Numerous new biosensor platforms are announced each year as researchers look for various electrode materials. Additionally, the researchers should familiarize themselves with the practical methods used in electrode production in order to build a successful biosensor. This review highlights current developments in modifying enzyme-immobilized biosensors for detection of different biomolecules. The article further describes methods of enzyme immobilization and various enzymes with different nanomaterials for the development of novel enzymatic electrochemical biosensors.

P66Shc Protein—Oxidative Stress Sensor or Redox Enzyme: Its Potential Role in Mitochondrial Metabolism of Human Breast Cancer

This work presents a comprehensive evaluation of the role of p66Shc protein in mitochondrial physiology in MDA-MB-231 breast cancer cells. The use of human breast cancer cell line MDA-MB-231 and its genetically modified clones (obtained with the use of the CRISPR-Cas9 technique), expressing different levels of p66Shc protein, allowed us to demonstrate how the p66Shc protein affects mitochondrial metabolism of human breast cancer cells. Changes in the level of p66Shc (its overexpression, and overexpressing of its Serine 36-mutated version, as well as the knockout of p66Shc) exert different effects in breast cancer cells. Interestingly, knocking out p66Shc caused significant changes observed mostly in mitochondrial bioenergetic parameters. We have shown that an MDA-MB-231 (which is a strong metastatic type of breast cancer) clone lacking p66Shc protein is characterized by a significant shift in the metabolic phenotype in comparison to other MDA-MB-231 clones. Additionally, this clone is significantly more vulnerable to doxorubicin treatment. We have proved that p66Shc adaptor protein in human breast cancer cells may exert a different role than in noncancerous cells (e.g., fibroblasts).

Mechanisms of muscle tissue adaptation in response to the influence of low-dose ionizing radiation

Mechanisms of interaction of ionizing radiation with biological objects are a chain of successive physical and physico-chemical changes, which manifest themselves in the form of excitation, primary and secondary ionization of molecules. Biosynthesis of ATP, which is carried out by a system of oxidation-reduction enzymes localized in the inner membrane of mitochondria - the respiratory chain, belongs to the vital processes that are directly disturbed under the action of ionizing radiation. The high degree of damage to this system is due to the significant radiosensitivity of metal-containing enzymes. The purpose of the work is to study the formation of the adaptive response of muscle tissue of sexually mature rats to the influence of ionizing radiation at a dose of 0.5 Gy. It was concluded that a day after irradiation with the dose of 0.5 Gy, the content of contractile proteins in skeletal muscle decreases slightly. On the 15th day, the content of contractile proteins began to decrease. By the 30th day, the content of contractile proteins decreased by 22.9% for myosin, by more than 11% for actin, and by 7 and 8% for troponin and tropomyosin, respectively, compared to the values of the intact group. A similar picture is observed in the cardiac muscle. Mg2+,Ca2+-ATP-ase activity of actomyosin, starting from the 1st day was shown to be increased in both the skeletal and cardiac muscles, reaching its peak in the cardiac muscle on the 15th day, in contrast to the skeletal muscle, where this indicator reached its peak on the 7th day. The authors conclude that irradiation of sexually mature animals with the dose of 0.5 Gy forms an adaptive response that is accompanied by an increase in Mg2+,Ca2+-ATP-ase activity due to the formation of a strong form of binding between F-actin and myosin, actin monomers go into the typical for actomyosin “turned on stage”, and the myosin heads acquire an ordered orientation in the muscle fiber. According to author’s idea, the data obtained indicated the benefit and reasonability of using in post-radiation dysfunctions complex pharmacological treatment drugs that are able to normalize intracellular homeostasis, eliminate probable acidic changes initiated by radiation exposure, activate the processes of intramuscle energy generation and which have protective properties in relation to the muscular system.

PerR: A Peroxide Sensor Eliciting Metal Ion-dependent Regulation in Various Bacteria.

Bacteria have to thrive in difficult conditions wherein their competitors generate partially reduced forms of oxygen, like hydrogen peroxide and superoxides. These oxidative stress molecules can also arise from within via the autoxidation of redox enzymes. To adapt to such conditions, bacteria express detox enzymes as well as repair proteins. Transcription factors regulate these defenses, and PerR is one of them. PerR is a Fur family transcriptional regulator that senses peroxide stress. Metal-bound PerR (either Mn2+ or Fe2+) can repress transcription of its regulon, but only the Fe2+-bound form of PerR can sense H2O2. This review describes different aspects of PerR and its varied roles, specifically in bacterial pathogens. Despite having roles beyond sensing peroxides, it is an underrated regulator that needs to be explored more deeply in pathogens.

Comparative Analysis of Ligninolytic Potential among Pleurotus ostreatus and Fusarium sp. with a Special Focus on Versatile Peroxidase

Lignocellulosic biomass is contemplated to be an inexpensive and copious feedstock that can be used for numerous industrial applications. However, lignin forms the lignin sheath and provides a physical barrier to enzymatic hydrolysis. In addition, lignin physically blocks cellulase, preventing it from being combined with the substrate in a process known as non-productive binding. Therefore, the depletion of lignin is a crucial method for obtaining fermentable sugars from the lignocellulosic biomass. Different white-rot fungi secrete different sets of lignin-mineralizing enzymes and each fungus secretes one or more of the three enzymes essential for lignin degradation. Among efficient redox enzymes, versatile peroxidase is extensively studied for its ability to degrade aromatics without the need for a mediator or polyvalent catalytic site. However, the presence of versatile peroxidase in F. spp. has not been studied. This study was planned with the objective of screening and comparing the production of versatile peroxidase enzymes from F. spp. and a standard culture of Pleurotus ostreatus MTCC-142. These fungal strains were first screened on solid media containing tannic acid, malachite green, or bromocresol green. The potency index for the tannic acid, malachite green, and bromocresol green on the 16th day of incubation was reported to be 1.28, 1.07, 1.09, and 1.10, respectively. Versatile peroxidase production patterns were investigated under solid state fermentation conditions for a period of 25 days at different temperatures ranging from 10 to 35 °C. The highest versatile peroxidase activity (592 UL−1) in F. sp. was observed at 30 °C after the 7th day of incubation. The molecular confirmation showed the presence of the vp gene in F. sp. along with Pleurotus ostreatus MTCC-142. The results determined that F. sp. possesses a versatile peroxidase enzyme and is able to degrade lignin efficiently, and thus it could be utilized as an alternative to other ligninolytic enzyme-producing fungi.

Smart biomaterials based on Laponite®: acute toxicity and influence on the redox state and enzyme activity in the organs of the detoxification system of mice

Smart biomaterials based on Laponite®: acute toxicity and influence on the redox state and enzyme activity in the organs of the detoxification system of mice

Full-length structure and heme binding in the transcriptional regulator HcpR.

HcpR is a CRP-family transcriptional regulator found in many Gram-negative anaerobic bacteria. In the perio-pathogen Porphyromonas gingivalis, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of the redox enzyme Hcp. However, the molecular mechanisms of heme binding to HcpR remain unknown. In this study we present the 2.3Å structure of the P. gingivalis HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from other family members. Using docking studies, we identify a putative heme binding site in the sensing domain. In vitro complementation and mutagenesis studies verify Met68 as an important residue in activation of HcpR. Finally, heme binding studies with purified forms of recombinant HcpR support Met68 and His149 residues as important for proper heme coordination in HcpR.

X-ray-based technologies in emerging fuel cell research

The workshop titled `X-ray-based technologies in emerging fuel cell research', organized by Vivian Stojanoff from Brookhaven National Laboratory (BNL) and Narayanasami Sukumar from Cornell University/Advanced Photon Source-Northeastern Collaborative Access Team, was a notable segment of the National Synchrotron Light Source II and Center for Functional Nanomaterials Users' Meeting held 13–17 May 2024. This one-day event, on 13 May 2024, at BNL in New York, aimed to bring together researchers, beamline scientists, management and developers to propel fuel cell technology forward using model systems inspired by natural photosynthesis and redox enzymes. This summary encapsulates the key discussions, advancements and future implications of the workshop.

Genome-wide survey of KT/HAK/KUP genes in the genus Citrullus and analysis of their involvement in K+-deficiency and drought stress responses in between C. lanatus and C. amarus

BackgroundThe KT/HAK/KUP is the largest K+ transporter family in plants, playing crucial roles in K+ absorption, transport, and defense against environmental stress. Sweet watermelon is an economically significant horticultural crop belonging to the genus Citrullus, with a high demand for K+ during its growth process. However, a comprehensive analysis of the KT/HAK/KUP gene family in watermelon has not been reported.Results14 KT/HAK/KUP genes were identified in the genomes of each of seven Citrullus species. These KT/HAK/KUPs in watermelon were unevenly distributed across seven chromosomes. Segmental duplication is the primary driving force behind the expansion of the KT/HAK/KUP family, subjected to purifying selection during domestication (Ka/Ks < 1), and all KT/HAK/KUPs exhibit conserved motifs and could be phylogenetically classified into four groups. The promoters of KT/HAK/KUPs contain numerous cis-regulatory elements related to plant growth and development, phytohormone response, and stress response. Under K+ deficiency, the growth of watermelon seedlings was significantly inhibited, with cultivated watermelon experiencing greater impacts (canopy width, redox enzyme activity) compared to the wild type. All KT/HAK/KUPs in C. lanatus and C. amarus exhibit specific expression responses to K+-deficiency and drought stress by qRT-PCR. Notably, ClG42_07g0120700/CaPI482276_07g014010 were predominantly expressed in roots and were further induced by K+-deficiency and drought stress. Additionally, the K+ transport capacity of ClG42_07g0120700 under low K+ stress was confirmed by yeast functional complementation assay.ConclusionsKT/HAK/KUP genes in watermelon were systematically identified and analyzed at the pangenome level and provide a foundation for understanding the classification and functions of the KT/HAK/KUPs in watermelon plants.

Antioxidant Carbon Dots Nanozymes Alleviate Stress-induced Depression by Modulating Gut Microbiota.

Depression is a debilitating mental illness that severely threatens millions of individuals and public health. Because of the multifactorial etiologies, there is currently no cure for depression; thus, it is urgently imperative to find alternative antidepressants and strategies. Growing evidence underscores the prominent role of oxidative stress as key pathological hallmarks of depression, making oxidative stress a potential therapeutic target. In this study, we report a N-doped carbon dot nanozyme (CDzyme) with excellent antioxidant capacity for treating depression by remodeling redox homeostasis and gut microbiota. The CDzymes prepared via microwave-assisted fast polymerization of histidine and glucose exhibit superior biocompatibility. Benefiting from the unique structure, CDzymes can provide abundant electrons, hydrogen atoms, and protons for reducing reactions, as well as catalytic sites to mimic redox enzymes. These mechanisms collaborating endow CDzymes with broad-spectrum antioxidant capacity to scavenge reactive oxygen and nitrogen species (•OH, O2-•, H2O2, ONOO-), and oxygen/nitrogen centered free radicals. A depression animal model was established by chronic unpredictable mild stress (CUMS) to evaluate the therapeutic efficacy of CDzymes from the behavioral, physiological, and biochemical index and intestinal flora assessments. CDzymes can remarkably improve depression-like behaviors and key neurotransmitters produced in hippocampus tissues and restore the gut microbiota compositions and the amino acid metabolic functions, proving the potential in treating depression through the intestinal-brain axis system. This study will facilitate the development of intestinal flora dysbiosis nanomedicines and treatment strategies for depression and other oxidative stress related multifactorial diseases.

The impact of the carbohydrate-binding module on how a lytic polysaccharide monooxygenase modifies cellulose fibers

BackgroundIn recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM “immobilizes” the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited.ResultsHere, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core.ConclusionsThese results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.

ВЛИЯНИЕ АГРОТЕХНОЛОГИИ ВОЗДЕЛЫВАНИЯ ГОРОХА НА БИОЛОГИЧЕСКИЕ И АГРОХИМИЧЕСКИЕ СВОЙСТВА ЛУГОВО-ЧЕРНОЗЕМНОЙ ПОЧВЫ

The aim of research is to establish patterns of changes in biological and agrochemical parameters of meadow-chernozem soil with long-term use of resource-saving treatments and chemicalization means in the conditions of the southern forest-steppe of Western Siberia. Stationary studies were conducted in the forest-steppe of the Omsk Region in a grain-fallow crop rotation (fallow – wheat – peas – wheat – barley) under a pea crop at the Omsk Agricultural Research Center. The parameters of changes in the number of individual physiological groups of microorganisms and indicators of biological activity of meadow-chernozem soil under peas were established using agricultural technologies with different levels of intensification, including the minimum-zero tillage system in crop rotation. It was determined that when using complex chemicalization agents in combination with No-till technology of soil cultivation, the activity of bacteria capable of fixing atmospheric nitrogen increased by 34 %, saprotrophic and amylolytic microorga¬nisms – by 13 and 17 % in relation to the control, respectively. The activity of the oxidation-reduction enzyme catalase when using intensification agents, regardless of the technology of pea cultivation, decreased to 28 % of the control. The use of intensification agents led to an increase in the content of nitrate nitrogen in the soil during the budding period, mobile phosphorus by 4 or more times. When assessing the anthropogenic impact on soil microflora, we used the definition of total biological activity, including all the studied biological tests in relative percentages. According to this indicator, soil cultivation options are arranged in a row in ascending order: combined &lt; moldboard &lt; minimum-zero.