Enzymatic Properties Research Articles

Biochemical characterization of Mycobacterial RNA polymerases.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), perform the essential function of RNA synthesis, we performed a series of in vitro transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.

Molecular cloning, expression, and biochemical characterization of carotenoid cleavage dioxygenase 1 (LbCCD1) from Lycium barbarum

Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, which have significant industrial applications due to their aromatic and bioactive properties. This study focuses on the molecular cloning and biochemical characterization of the LbCCD1 from Lycium barbarum. The LbCCD1 gene was successfully cloned and heterologously expressed in Escherichia coli and several carotenoids were using as substrates for investigate its specificity by in vitro and in vivo experiment. The LbCCD1 protein was able to cleave a variety of carotenoids including β-carotene, zeaxanthin, astaxanthin, and β-apo-8′-carotenal, at the 9, 10 (9′, 10′) double bond to produce β-ionone, 3‑hydroxy-4-oxo-β-ionone, and 3‑hydroxy-β-ionone, respectively in vitro. LbCCD1 could also cleave zeaxanthin and β-carotene at the 9, 10 (9′, 10′) double bond to produce β-ionone, respectively, in E. coli accumulating carotenoids. Interestingly, LbCCD1 did not exhibit cleavage activity on lycopene either in vivo or in vitro unlike other CCD1 family enzymes.In the previous experiment, it was confirmed that LbCCD1 exhibits cleavage activity towards β-apo-8′-carotenal in vitro, so we used β-apo-8′-carotenal as the substrate for characterizing the enzymatic properties. The expression of LbCCD1 was optimized at such conditions (temperature 24 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of LbCCD1 revealed the optimal activities were at pH 9 and 55 °C. The addition of 10 % ethanol could increase enzyme activity to above 15 %. However, the concentration of Fe2+ has a minimal effect on enzyme activity. The Vmax for β-apo-8′-carotenal was 8.6 U/mg, while the Km was 0.27 mM. To preliminarily verify the potential of LbCCD1 as a biological component for β-ionone production. By introducing LbCCD1 into the β-carotene-high-producing chassis cell and optimizing the conditions (temperature 30 °C, IPTG 0.01 mM, Fe2+ concentration 0.05 mM), the β-ionone yield reached 21.45 mg/L. This study focused on one of the CCDs derived from woody plants, which have been relatively underexplored. It lays the groundwork for expanding the CCD enzyme library, identifying suitable CCDs, and investigating the structure-function relationship of CCDs. Furthermore, it sets the stage for engineering novel CCD genes and developing advanced applications of CCDs as biocatalysts and platforms for synthetic biology. These advancements will enable the efficient production of volatile aroma compounds from carotenoids.

Intra-articular injection of platinum nanozyme-loaded silk fibroin/pullulan hydrogels relieves osteoarthritis through ROS scavenging and ferroptosis suppression

Reactive oxygen species (ROS)-mediated ferroptosis plays a critical role in the development of osteoarthritis (OA). Consequently, it is speculated that anti-ferroptosis agents could represent a novel therapeutic strategy for managing OA. In this study, a hydrogel incorporating platinum (Pt) nanozyme was synthesized by dispersing Pt nanoparticles (NPs) within a matrix of silk fibroin (SF) and oxidized pullulan (oxPL). This hydrogel allows for a substantial and sustained release of up to 30 days. The gelation time (from 140.3 ± 42.3 s to 460.0 ± 40.0 s), swelling capacity (from 57.7 ± 3.8 % to 24.0 ± 7.0 %), and degradation rate (from 60.3 ± 4.7 % to 32.0 ± 4.6 %) of the hydrogels can be modulated by adjusting the Pt NP content. The Pt@SF/oxPL hydrogel effectively eliminates ROS due to its catalase-like and superoxide dismutase-like enzymatic properties. In vitro studies demonstrated that Pt@SF/oxPL efficiently mitigated the process of ferroptotic cell death in chondrocytes. More critically, intra-articular administration of Pt@SF/oxPL showcased therapeutic advantages by both protecting and stimulating the regeneration of cartilage throughout the progression of OA. Collectively, this study suggests that Pt@SF/oxPL hydrogels could potentially serve as an effective treatment for OA, presenting a novel nanozyme-based therapeutic approach for this condition.

Glycine as a stabilizing osmolyte for Renilla luciferase: A kinetic and molecular dynamics analysis

Renilla luciferase, a luminescent enzyme, is utilized in gene expression analysis and biosensor technology, and extensive research has been conducted on its structure and function. Glycine is an osmolyte that plays a key role in protein stabilization against denaturation. However, its impact on enzyme properties is unpredictable. This study aimed to investigate the effect of glycine on the kinetics and stability of Renilla luciferase. The data revealed that glycine at a concentration range of 0.1 to 0.6 M improved enzyme kinetics and thermal stability. The highest catalytic efficiency was observed at a concentration of 0.5 M. Molecular dynamics simulations demonstrated that in the presence of 0.5 M glycine, substrate access to the enzyme’s active site was enhanced while the root-mean-square fluctuation (RMSF) of the protein backbone was reduced. Additionally, the analysis of protein-water hydrogen bonding interactions showed an increase in the hydrogen bonding between water molecules and Renilla luciferase. The present study may be used for the formulation of Renilla luciferase for commercial purposes.

Zonulin — effective biomarker for the diagnosis of intestinal lesions in patients with nonalcoholic fatty liver disease in COVID‑19

The SARS‑CoV‑2 virus mainly attacks the respiratory system, but can also affect several organs, in particular, causing gastrointestinal symptoms such as vomiting, diarrhea or abdominal pain in the early stages of the disease. Intestinal dysfunction leads to a change in intestinal microorganisms and an increase in the content of inflammatory cytokines. Liver dysfunction is also relatively common in patients with coronavirus disease 2019 (COVID19). Objective — to investigate the efficacy of zonulin in determining intestinal damage in patients with nonalcoholic fatty liver disease with COVID‑19. Materials and methods. The study included 76 patients with non‑alcoholic fatty liver disease (NAFLD) who had complaints indicating intestinal disorders during the outpatient stage of follow‑up after ­COVID‑19. From the anamnesis, it was found that all examined patients had a confirmed diagnosis of infection with SARS‑CoV‑2 virus. All patients had a mostly mild course of COVID‑19, which did not require hospitalization. Patients with NAFLD were divided into two groups depending on defecation violation: group I — 36 patients who, after an acute respiratory infection caused by the SARS‑CoV‑2 virus, developed periodic diarrhea (on average, in (38.7±5.2) days after a negative PCR result for SARS‑CoV‑2 virus); group II — 40 patients with constipation, which began to bother on average in (86.9±7.4) days after a negative PCR result. The control group included 20 practically healthy individuals. The level of zonulin was determined in blood serum and faeces by enzyme‑linked immunosorbent assay. Patients underwent faecal culture to assess the quantitative and qualitative composition of the colon microflora. Results. During the anthropometric examination, overweight and first‑degree obesity were detected more frequently in patients of both groups: overweight was diagnosed most frequently in patients of group I (38.9%) and first‑degree obesity in patients of group II (35.0%). The microbiological examination of faeces in both groups of patients revealed a decrease in the number of bifidus and lactobacilli, as well as Enterococcus and E. coli with normal enzymatic properties. In group I patients, Citrobacter and Staphylococcus were statistically significantly (p <0.05) more often detected in increased concentrations in faeces, whereas in group II patients Enterobacter, Klebsiella and Clostridium were detected (p <0.05). Significant changes were found in the analysis of zonulin levels in both serum and faeces in patients with NAFLD after COVID‑19. Serum zonulin levels in patients of group I increased 6.5‑fold, and in patients of group II—8.6‑fold (p <0.001). An increase in the concentration of zonulin in the faeces was also detected — by 7.5 and 9.7 times, respectively (p <0.001). A correlation was established between the level of zonulin in both blood serum and faeces and a decrease in the number of bifidus and lactobacilli in faeces in patients of both groups. Also, a strong correlation was found between the number of Enterobacter, Klebsiella and Clostridium and a medium intensity correlation between the number of Staphylococcus in the faeces in patients of group ІІ. In patients of group І, a correlation was found between the number of Staphylococcus and Citrobacter. The level of zonulin (both in blood serum and faeces) directly depended on changes in body mass index in the patients we examined, namely, overweight in patients of group І and obesity of the first degree in patients of group ІІ. Conclusions. In patients with NAFLD after COVID19, changes in the quantitative and qualitative composition of the colon microflora were found in the intestinal lesions, including a decrease in the number of bifidobacteria and lactobacilli, as well as an increase in the content of Staphylococcus and Citrobacter in the faeces in patients with predominant diarrhoea and Enterobacter, Klebsiella and Clostridium in patients with predominant constipation. An increase in the level of zonulin in the blood serum and faeces in patients with NAFLD after ­COVID‑19 was detected. A more pronounced deviation from the norm was found in patients of group ІІ (with complaints of constipation). A direct correlation was established between the level of zonulin in the blood serum and faeces and changes in the quantitative and qualitative composition of the colon microflora, as well as an increase in body mass index in patients with NAFLD after ­COVID‑19.

Catalytic Potential-Guided Design of Multi-Enzymatic System for DHA Production from Glycerol

The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni+2 agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 °C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations.

PLS‐guided Mutant Recombination to Improve the Stability of Bovine Enterokinases Obtained by Directed Evolution

Activity and thermostability are critical yet challenging to improve simultaneously in enzymes. Using directed evolution, we previously identified bovine enterokinase (EKL) variants with enhanced soluble expression and thermal stability. Partial least squares (PLS) analysis of 321 EKL variants revealed the impact of individual mutations and identified neutral or detrimental mutations in top‐performing variants. Leveraging PLS rankings, we created new variants with fewer mutations and enhanced stability. Most original and PLS‐guided variants exhibited an activity‐stability trade‐off. However, two new triple‐ and quadruple‐mutants improved both activity and stability, surpassing the trade‐off limit. Recombining PLS‐guided mutations likely eliminated neutral or harmful mutations, enhancing stability. MD simulations linked residue‐specific dynamics with stability, pinpointing critical structural regions near aggregation‐prone areas. Our findings validate PLS as a potent strategy to enhance enzyme properties, complementing directed evolution.

Partially Bio-Based and Biodegradable Poly(Propylene Terephthalate-Co-Adipate) Copolymers: Synthesis, Thermal Properties, and Enzymatic Degradation Behavior.

A series of partially bio-based and biodegradable poly(propylene terephthalate-co-adipate) (PPTA) random copolymers with different components were prepared by the melt polycondensation of petro-based adipic acid and terephthalic acid with bio-based 1,3-propanediol. The microstructure, crystallization behavior, thermal properties, and enzymatic degradation properties were further investigated. The thermal decomposition kinetics was deeply analyzed using Friedman's method, with the thermal degradation activation energy ranging from 297.8 to 302.1 kJ/mol. The crystallinity and wettability of the copolymers decreased with the increase in the content of the third unit, but they were lower than those of the homopolymer. The thermal degradation activation energy E, carbon residue, and reaction level n all showed a decreasing trend. Meanwhile, the initial thermal decomposition temperature (Td) was higher than 350 °C, which can meet the requirements for processing and use. The PPTA copolymer material still showed excellent thermal stability. Adding PA units could regulate the crystallinity, wettability, and degradation rate of PPTA copolymers. The composition of PPTA copolymers in different degradation cycles was characterized by 1H NMR analysis. Further, the copolymers' surface morphology during the process of enzymatic degradation also was observed by scanning electron microscopy (SEM). The copolymers' enzymatic degradation accorded with the surface degradation mechanism. The copolymers showed significant degradation behavior within 30 days, and the rate increased with increasing PA content when the PA content exceeded 45.36%.

Sensitive colorimetric detection of heparin using reverse modulation of peroxidase- and oxidase-mimetic activities in Fe3O4@PDA@MnO2 nanocomposites

Heparin, a widely studied glycosaminoglycan, plays crucial roles in the regulation of various physiological and pathological processes. Therefore, it's important to develop highly selective and sensitive methods for convenient monitoring of heparin levels in biological systems. We report the design and synthesis of Fe3O4@PDA@MnO2 nanoparticles (FPM-NPs), which exhibit dual enzymatic activities, enabling quantitative detection of heparin. The FPM-NPs feature a unique tri-layer spherical shell structure, possessing both peroxidase-like and oxidase-like activities, and catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence or absence of H2O2. Remarkably, upon co-incubated with heparin, the oxidase activity of FPM-NPs decreases, while the peroxidase activity increases. By leveraging these dual enzymatic properties of FPM-NPs, a highly sensitive and specific colorimetric detection of heparin is achieved, with a detection limit reaching 6.51 nM and a good linear response to quantify heparin ranging 10–800 nM. Additionally, the developed FPM-NPs are successfully applied to measure heparin in fetal bovine serum samples. We also extend this detection method to a paper-based chip, enabling portable detection of heparin through grayscale analysis of mobile phone photographs. The multi-nanozyme-based heparin detection approach provides a new perspective for future research on expanding the application of nanocomposite materials in biomedical detection and analysis.

Identification of a transglutaminase from Bacillus amyloliquefaciens: Gene mining, protein expression, mechanism analysis and enzymatic characterization

Transglutaminase (TGase) can catalyze the crosslinking within or between protein molecules, improving the structural and functional properties of proteins. It has been widely used in artificial meat, yogurt products, flour products and other foods. In this study, a high yield TGase strain was screened from fermented soybean, and it was identified as the Bacillus amyloliquefaciens XCT-09. Then, the transglutaminase (X9Tgl) from XCT-09 was expressed in Escherichia coli BL21(DE3), and the enzyme activity of purified X9Tgl reached 5.73 U/mg. Subsequently, the catalytic mechanism of X9Tgl was elucidated, and the X9Tgl contained the active center of E115, C116 and E187, as well as the substrate binding sites W149 and F69. Furthermore, this study characterized the enzymatic properties of X9Tgl, and the optimal catalytic temperature and pH were 60 °C and 6, respectively. Moreover, X9Tgl exhibited stronger thermal stability and pH tolerance than STG, and it was not inhibited by the majority of metal ions. This study successfully identified a X9Tgl with high thermal stability, and pH tolerance, which provided a potential TGase resource for application in the food and medicine fields.

Genome-guided bioprospecting for industrially important enzymes from a thermophilic Bacillus subtilis strain SR-7, an isolate from hot spring of Madhya Pradesh, India

The study focused on isolating thermophilic bacteria from the hot springs of Madhya Pradesh, India, with the aim of identifying strains capable of producing industrially valuable enzymes. Among the 15 isolated strains, SR-7 identified as Bacillus subtilis exhibited significant enzyme activity, leading to its selection for further investigation. Genomic analysis revealed key characteristics of SR-7, including a 3,593,163 base pair chromosome with a GC content of 44.14 %. The genome was found to contain 55 tRNA genes, four rRNA clusters, and 3744 protein-coding genes. These genes encompass essential metabolic pathways and genes responsible for thermophilic adaptation, highlighting the potential for industrial applications. Further experimentation demonstrated SR-7 have the ability to produce various enzymes of industrial significance, such as amylase, lipase, cellulase, protease, lecithinase, gelatinase, and pectinase. Importantly, enzyme production was observed across a broad pH range (6.5 to 10) and temperature spectrum, indicating the versatility and adaptability of SR-7 to different environmental conditions. The findings suggest that SR-7 holds promise as a valuable resource for the pharmaceutical and industrial sectors. Its capacity to produce thermostable enzymes suitable for a range of applications underscores its potential contribution to biotechnological processes. By harnessing the enzymatic capabilities of thermophilic bacteria like SR-7, industries can pursue more sustainable and efficient production methods, reducing reliance on traditional chemical processes and enhancing overall productivity. Further research into the enzymatic properties and genetic mechanisms of SR-7 may unveil additional avenues for its utilization in various industrial processes.

Unlocking Lignin's Potential: Engineered Bacterial Laccases to Produce Biologically Active Molecules.

Laccases are biocatalysts with immense potential in lignocellulose biorefineries to valorize emerging lignin monomers for sustainable chemicals. Despite reduced costs over the past two decades, enzymes remain a major expense in biorefining. Protein engineering can enhance enzyme properties and lower costs further. In this study, we used enzyme engineering tools to improve > 400-fold the catalytic efficiency (kcat/Km) of a hyperthermostable bacterial laccase for 2,6-dimethoxyphenol, a lignin-related phenolic compound. Furthermore, this evolved variant showed improved activity at neutral to alkaline pH for hydroxycinnamyl alcohols, hydrocinnamic acids, phenylpropanoid and vanillyl derivatives. We optimized conditions for the synthesis of syringaresinol, dehydrodiconiferyl alcohol, thomasidioic acid, biseugenol, dehydrodiisoeugenol, and diapocynin, detailing the pH, catalyst concentration, reaction time, temperature, and oxygenation of the reaction mixtures. Our biocatalytic system offers several advantages, including being free of organic solvents, achieving faster reaction times, using lower amounts of enzymes and delivering excellent yields (up to 100%) than reported methods. Additionally, we provide insights that advance the state-of-the-art in lignin combinatory chemistry. This progress marks a significant step forward in valorizing the lignin chemicals platform, enabling high yields of dimeric compounds with structural scaffolds that can be exploited in various biotechnological areas, such as medicinal chemistry and polymer synthesis.

Heterologous expression of GH11 xylanase from Myceliophthora heterothallica F.2.1.4 in Pichiapastoris

This research is centered on improving a xylanase enzyme derived from Myceliophthora heterothallica by expressing it in Pichia pastoris to enhance biomass hydrolysis. Lignocellulosic biomass holds significant potential for renewable applications in biofuels, chemicals, and pharmaceuticals. The study explores the impact of the choice of expression host on enzyme properties, specifically addressing challenges in thermostability by utilizing P. pastoris and leveraging its glycosylation capabilities. Previously expressed in Escherichia coli, the xylanase exhibited in this work cooperative kinetics, pH stability, and resistance to phenolic compounds. Gene integration and expression in P. pastoris were verified through PCR and activity assays. After 120 h of induction, an enzymatic activity of 48.8 U mL−1 was obtained. Subsequent characterization revealed improved specific activity, substrate affinity, and optimal temperature compared to the enzyme expressed in E. coli. The enzyme exhibited excellent pH and temperature stability for industrial applications, maintaining over 90% of its activity within a pH range of 5.0–10.0 and remaining stable even after 90 min of incubation at 55 °C. It also demonstrated resistance to metal ions and responsiveness to phenolic compounds. These findings underscore the versatility of the recombinant xylanase from M. heterothallica expressed in P. pastoris, highlighting its potential as a valuable resource for biomass conversion. The study emphasizes the pivotal role of host choice in optimizing enzyme characteristics for industrial applications, underscoring the importance of employing heterologous expression systems, as demonstrated in this investigation.

Sequence analysis, homology modeling, tissue expression, and potential functions of seven putative acetylcholinesterases in the spider Cupiennius salei.

Acetylcholine esterases (AChEs) are essential enzymes in cholinergic synapses, terminating neurotransmission by hydrolysing acetylcholine. While membrane bound AChEs at synaptic clefts efficiently perform this task, soluble AChEs are less stable and effective, but function over broader areas. In vertebrates, a single gene produces alternatively spliced forms of AChE, whereas invertebrates often have multiple genes, producing both enzyme types. Despite their significance as pesticide targets, the physiological roles of invertebrate AChEs remain unclear. Here, we characterized seven putative AChEs in the wandering spider, Cupiennius salei, a model species for neurophysiological studies. Sequence analyses and homology modeling predicted CsAChE7 as the sole stable, membrane-bound enzyme functioning at synaptic clefts, while the others are likely soluble enzymes. In situ hybridization of sections from the spider's nervous system revealed CsAChE7 transcripts co-localizing with choline acetyltransferase in cells that also exhibited AChE activity. CsAChE7 transcripts were also found in rapidly adapting mechanosensory neurons, suggesting a role in precise and transient activation of postsynaptic cells, contrasting with slowly adapting, also cholinergic, neurons expressing only soluble AChEs, which allow prolonged activation of postsynaptic cells. These findings suggest that cholinergic transmission is influenced not only by postsynaptic receptors but also by the enzymatic properties regulating acetylcholine clearance. We also show that acetylcholine is a crucial neurotransmitter in the spider's visual system and sensory and motor pathways, but absent in excitatory motor neurons at neuromuscular junctions, consistent with other arthropods. Our findings on sequence structures may have implications for the development of neurological drugs and pesticides.

Deep Learning-Based Self-Adaptive Evolution of Enzymes

AbstractBiocatalysis has been widely used to prepare drug leads and intermediates. Enzymatic synthesis has advantages, mainly in terms of strict chirality and regional selectivity compared with chemical methods. However, the enzymatic properties of wild-type enzymes may or may not meet the requirements for biopharmaceutical applications. Therefore, protein engineering is required to improve their catalytic activities. Thanks to advances in algorithmic models and the accumulation of immense biological data, artificial intelligence can provide novel approaches for the functional evolution of enzymes. Deep learning has the advantage of learning functions that can predict the properties of previously unknown protein sequences. Deep learning-based computational algorithms can intelligently navigate the sequence space and reduce the screening burden during evolution. Thus, intelligent computational design combined with laboratory evolution is a powerful and potentially versatile strategy for developing enzymes with novel functions. Herein, we introduce and summarize deep-learning-assisted enzyme functional adaptive evolution strategies based on recent studies on the application of deep learning in enzyme design and evolution. Altogether, with the developments of technology and the accumulation of data for the characterization of enzyme functions, artificial intelligence may become a powerful tool for the design and evolution of intelligent enzymes in the future.

Immobilization of Lipase from Thermomyces Lanuginosus and Its Glycerolysis Ability in Diacylglycerol Preparation.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

Analysis of changes in intestinal microbiota in female Wistar rats at the stage of breast cancer induction by N-methyl-N-nitrosourea

The intestinal microbiota, having enormous metabolic potential, makes a significant contribution to the physiological and pathological processes of humans and animals and is currently considered as an important factor in the pathogenesis of cancer. The aim of this study is to determine changes in the quantitative and qualitative composition of the intestinal microbiota in Wistar rats during chemical induction of breast cancer (BC). Material and methods. The work was performed on female Wistar rats (n = 40) aged 3 months, weighing 200–210 g, using cultural methods for studying fecal microbiota in intact rats (1 group) on the 1st, 14th, 35th days and in rats with induction of breast cancer and and in rats, whereby N-methyl-N-nitrosourea was administered to induce breast cancer (2 group) on the 1st (before injection of N-methyl-N-nitrosourea), 14th, 35th days after injection of N-methyl-N-nitrosourea. Results and discussion. In all experimental animals, representatives characteristic of the intestinal normobiota of warm-blooded animals predominated, namely: Bifidobacterium spp., Lactobacillus spp., Escherichia coli with pronounced enzymatic properties, Enterococcus spp., Clostridium spp. In addition, Staphylococcus spp., yeast-like fungi of the genus Candida and mold. Escherichia coli with reduced enzymatic activity was also detected. It was established that the isolated bacteria belonged to 3 types, 4 classes, 5 orders, 6 families, 6 genera of the bacterial domain. Also, 2 genera of fungi belonging to the order Saccharomycetales were isolated. The most significant changes in the composition of the intestinal microbiota were noted in rats with chemically induced breast cancer on the 35th day tumor induction: the appearance of pathogenic microflora in the intestine was revealed.

Hydrolysis Mechanism of Multimodular Endoglucanases with Distinctive Domain Composition in the Saccharification of Cellulosic Substrates.

Two multimodular endoglucanases in glycoside hydrolase family 5, ReCel5 and ElCel5, share 73% identity and exhibit similar modular structures: family 1 carbohydrate-binding module (CBM1); catalytic domain; CBMX2; module of unknown function. However, they differed in their biochemical properties and catalytic performance. ReCel5 showed optimal activity at pH 4.0 and 70 °C, maintaining stability at 70 °C (>80% activity). Conversely, ElCel5 is optimal at pH 3.0 and 50 °C (>50% activity at 50 °C). ElCel5 excels in degrading CMC-Na (256 U/mg vs 53 U/mg of ReCel5). Five domain-truncated (TM1-TM5) and four domain-replaced (RM1-RM4) mutants of ReCel5 with the counterparts of ElCel5 were constructed, and their enzymatic properties were compared with those of the wild type. Only RM1, with ElCel5-CBM1, displayed enhanced thermostability and activity. The hydrolysis of pretreated corn stover was reduced in most TM and RM mutants. Molecular dynamics simulations revealed interdomain interactions within the multimodular endoglucanase, potentially affecting its structural stability and complex biological catalytic processes.

Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry.

UV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA-and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo . We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid-protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

Double hook-type aptamer-based colorimetric and electrochemical biosensor enables rapid and robust analysis of EpCAM expression

Epithelial cell adhesion molecule (EpCAM), which is overexpressed in breast cancer cells and participates in cell signaling, migration, proliferation, and differentiation, has been utilized as a biomarker for cancer diagnosis and therapeutic prognosis. Here, a dual-signal readout nonenzymatic aptasensor is fabricated for the evaluation of EpCAM at the level of three breast cancer cell lines. The central principle of this enzyme-free aptasensor is the use of double hook-type aptamers (SYL3C and SJ3C2)-functionalized magnetic iron oxide (Fe3O4) as capture probes and quasi-CoFe prussian blue analogs (QCoFe PBAs) as nonenzymatic signal probes for colorimetric and electrochemical analysis. Following ligand detachment, the CoFe PBA was transformed to QCoFe PBA (calcined at 350 °C for 1 h), with its metal active sites exposed by controllable pyrolysis. We found that the enhanced sensitivity was attributed to the resonance effect of QCoFe PBA with the remarkable enzymatic properties. The dual-signal readout nonenzymatic aptasensor exhibited limits of detection for EpCAM as low as 0.89 pg mL−1 and 0.24 pg mL−1, within a wide linear range from 0.001 to 100 ng mL−1, respectively. We successfully employed this nonenzymatic aptasensor for monitoring EpCAM expression in three breast cancer cell lines, which provides an economical and robust alternative to costly and empirical flow cytometry. The dual-signal readout nonenzymatic aptasensor provides rapid, robust, and promising technological support for the accurate management of tumors.