Activation Of Enzyme Research Articles

Incidence of Pancreatic Leakage in Isolated Biliary Limb Roux-en-Y Reconstruction in Whipple Procedure

Abstract Background Whipple is currently the procedure of choice in the treatment of localized pancreatic cancer. Isolated Roux-en-Y loop pancreaticojejunostomy was proposed as a method of reconstruction following pancreaticoduodenectomy. The idea is to isolate the bile from pancreatic secretions to minimize pancreatic enzyme activation and ultimately pancreatic fistula formation. It was also proposed that if a pancreatic fistula develops, it will be a pure fistula and thereby more benign as opposed to a mixed pancreatic fistula containing bile. Objective To estimate the incidence of pancreatic fistula in patients undergoing isolated Roux-en- Y hepaticojejunostomy reconstruction following pancreaticoduodenectomy. Patients and Methods In this prospective observational study, our aim is to calculate the incidence of post-operative pancreatic fistula in 20 patients that underwent Roux-en-Y isolated biliary limb reconstruction following pancreaticoduodenectomy for the treatment of pancreatic cancer. The study includes patients that underwent this procedure at Ain Shams University Hospitals in the period between January 2021 and July 2022 with a post-operative follow up period of 3 weeks. Patients were diagnosed with pancreatic cancer using MRI or CT scans with some of them also undergoing endoscopic US with biopsy. They were then admitted to one of the two main Ain Shams University Hospitals for pre-operative preparation. Results Our study did no estimate post-operative complications such as delayed gastric emptying and wound infection. Pancreaticoduodenectomy is a major surgery that carries a significant burden in terms of morbidity and mortality. Post-operative pancreatic fistula can be fatal as it can lead to vascular pseudoaneurysms that may fatally rupture. The idea behind using isolated biliary limb roux-en-y reconstruction is to alleviate the morbidity caused by post-operative pancreatic fistula. Conclusion Further comparative studies with a higher number of patients are recommended to assess and compare the value of isolated biliary limb reconstruction when compared to conventional reconstruction.

An Enzyme-Responsive Self-Immolative Recognition Marker for Manipulating Cell-Cell Interactions.

The development of innovative strategies for cell membranes engineering is of prime interest to explore and manipulate cell-cell interactions. Herein, an enzyme-sensitive recognition marker that can be introduced on cell surface via bioorthogonal chemistry is designed. Once functionalized in this fashion, the cells gain the ability to assemble with cell partners coated with the complementary marker through non-covalent click chemistry. The artificial cell adhesion induces natural biological processes associated with cell proximity such as inhibiting cancer cell proliferation and migration. On the other hand, the enzymatic activation of the stimuli-responsive marker triggers the disassembly of cells, thereby restoring the tumor cell proliferation and migration rates. Thus, the study shows that the ready-to-use complementary markers are valuable tools for controlling the formation and the breaking of bonds between cells, offering an easy way to investigate biological processes associated to cell proximity.

Limits of Non‐invasive Enzymatic Activation by Local Temperature Control (Small 30/2024)

Limits of Non‐invasive Enzymatic Activation by Local Temperature Control (Small 30/2024)

A Functional Human Glycogen Debranching Enzyme Encoded by a Synthetic Gene: Its Implications for Glycogen Storage Disease Type III Management.

Glycogen Storage Disease type III (GSD III) is a metabolic disorder resulting from a deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (approximately 170 kDa) with cytoplasmic localization and two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Mutations in the Agl gene, with consequent deficiency in GDE, lead to the accumulation of abnormal/toxic glycogen with shorter chains (phosphorylase limit dextrin, PLD) in skeletal and/or heart muscle and/or in the liver. Currently, there is no targeted therapy, and available treatments are symptomatic, relying on specific diets. Enzyme Replacement Therapy (ERT) might represent a potential therapeutic strategy for GSD III. Moreover, the single-gene nature of GSD III, the subcellular localization of GDE, and the type of affected tissues represent ideal conditions for exploring gene therapy approaches. Toward this direction, we designed a synthetic, codon-optimized cDNA encoding the human GDE. This gene yielded high amounts of soluble, enzymatically active protein in Escherichia coli. Moreover, when transfected in Human Embryonic Kidney cells (HEK-293), it successfully encoded a functional GDE. These results suggest that our gene or protein might complement the missing function in GSD III patients, opening the door to further exploration of therapeutic approaches for this disease.

A distributionally robust optimization approach for the potassium fertilizer product transportation considering transshipment through crossdocks

Potassium fertilizer is an essential input for agricultural productivity, and plays a critical role in various plant processes, influencing water uptake, enzyme activation, and photosynthesis. The efficient delivery of potassium fertilizer products to the end-users, typically farmers and agricultural enterprises, is of utmost importance. Due to the long traveling distance between the supplier and customers, decision makers of supplier normally set up multiple crossdocks to aid in transition and storage of potassium fertilizer products. In this situation, making an optimal transportation plan as well as the inventory level of crossdocks will directly enhance the efficiency and effectiveness of long-distance transportation. In this study, we model this problem as a dynamic transportation problem with transshipment considering the uncertain time-varying customers’ demand. To characterize the demand information, we first construct an ambiguity set of customers’ demand based in limited historical data and then develop a distributionally robust optimization-based (DRO) framework to optimize the transportation plan and related inventory level of crossdocks simultaneously. We also propose a general approach to overcome the computational challenges of DRO by transforming the original DRO into a second-order cone programming based on duality theory. Additionally, we introduce linear decision rules to adjust the optimization strategy based on the new observed demand, thus lead the model to handle the dynamic information flow in real time. In case study, all involved data are collected or derived from a real potassium fertilizer company located at Western China. The results show our model reduces the cost by 18.07% compared to stochastic model (sample average approximation), indicating a significant effectiveness of our model on the improvement of delivery efficiency and cost saving in real dynamic logistic systems. Also, we conclude the managerial insight that decision-makers should develop a comprehensive strategy, including improving communication to ensure order status updates, planning rationally to evenly distribute orders, and proactively allocating resources to meet operational demands.

NOX-2 Inhibitors may be Potential Drug Candidates for the Management of COVID-19 Complications.

COVID-19 is an RNA virus that attacks the targeting organs, which express angiotensin- converting enzyme-2 (ACE-2), such as the lungs, heart, renal system, and gastrointestinal tract. The virus that enters the cell by endocytosis triggers ROS production within the confines of endosomes via a NOX-2 containing NADPH-oxidase. Various isoforms of NADPH oxidase are expressed in airways and alveolar epithelial cells, endothelial and vascular smooth muscle cells, and inflammatory cells, such as alveolar macrophages, monocytes, neutrophils, and Tlymphocytes. The key NOX isoform expressed in macrophages and neutrophils is the NOX-2 oxidase, whereas, in airways and alveolar epithelial cells, it appears to be NOX-1 and NOX-2. The respiratory RNA viruses induce NOX-2-mediated ROS production in the endosomes of alveolar macrophages. The mitochondrial and NADPH oxidase (NOX) generated ROS can enhance TGF-β signaling to promote fibrosis of the lungs. The endothelium-derived ROS and platelet-derived ROS, due to activation of the NADPH-oxidase enzyme, play a crucial role in platelet activation. It has been observed that NOX-2 is generally activated in COVID-19 patients. The post-COVID complications like pulmonary fibrosis and platelet aggregation may be due to the activation of NOX-2. NOX-2 inhibitors may be a useful drug candidate to prevent COVID-19 complications like pulmonary fibrosis and platelet aggregation.

Exploring the therapeutic potential of the oxygenated monoterpene linalool in alleviating saline stress effects on Allium cepa L.

Sodium chloride (NaCl) can cause oxidative stress in plants, which represents a potential obstacle to the development of monocultures worldwide. Onion (Allium cepa L.) is a famous vegetable consumed and used in world cuisine. In the present study, we analyzed the influence of soil physicochemical profile and the remedial capacity of linalool on seed emergence, roots, and leaf growth in onions subjected to salt stress, as well as its in vivo and in vitro antioxidant potential, Fe2+chelating activity, and reducing power of Fe3+. The outcome of the soil analysis established the following order of abundance: sulfur (S) > calcium (Ca) > potassium (K) > magnesium (Mg) > sodium (Na). NaCl (150 mM) significantly reduced the emergence speed index (ESI), leaf and root length, while increasing the peroxidation content. The length of leaves and roots significantly increased after treatment with linalool (300 and 500 μg/mL). Our data showed negative correlations between seed emergence and K+ concentration, which was reversed after treatments. Linalool (500 μg/mL) significantly reduced oxidative stress, but increased Fe2+ concentration and did not show potential to reduce Fe3+. The in vivo antioxidant effect of linalool is thought to primarily result from an enzymatic activation process. This mechanism underscores its potential as a therapeutic agent for oxidative stress-related conditions. Further investigation into this process could unveil new avenues for antioxidant therapy.

Directed Evolution of Protein-Based Sensors for Anaerobic Biological Activation of Methane.

Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives.

Peningkatan Mutu Kakao Melalui Percepatan Lama Fermentasi dengan Penggunaan Pektinase Aspergillus niger dan Ion Kalsium

Indonesia is the third largest producer of cocoa beans (Theobroma cacao L.) in the world, but the quality of Indonesian cocoa beans is considered as low. Fermentation is one of the factors that affect the quality of cocoa beans. Fermentation of cocoa beans is a spontaneous fermentation and takes about 5-7 days (for bulk cacao). The objective of this study was to determine the potential of adding pectinase from Aspergillus niger and calcium ions in accelerating fermentation time and the quality of cocoa beans produced. The independent variable in this study was the concentration of calcium ions as an enzyme activator. The concentration of calcium ions and pectinase are applied to cocoa fermentation. The observations made were reducing sugar content, total sugar content, protein content, lactic acid bacteria and acetic acid bacteria levels. Parametric data will be analysed by T-test independent with a confidence level of α=0.05. The results showed that added 4.5 units of pectinase and 100 mM calcium ions have significant in reduce the total sugar compared with control due to enzimatic activity. Therefore, the addition of pectinase enzymes from Aspergillus niger and calcium ions have a potential to accelerate the fermentation time and the fermented cocoa beans that have been carried out have met the quality standards of SNI 2828:2008 and included in I – B group of forastero cocoa. This potential needs to be tested further with a larger fermentation volume, for example above 40 kg, to achieve the optimal temperature for the formation of flavor precursors. Keywords: cocoa bean, ion calcium, pectinase, quality

Use of ultrasound to improve the activity of cyclodextrin glycosyltransferase in the producing of β-cyclodextrins: Impact on enzyme activity, stability and insights into changes on enzyme macrostructure

This work explored the impact of ultrasound (US) on the activity, stability, and macrostructural conformation of cyclodextrin glycosyltransferase (CGTase) and how these changes could maximize the production of β-cyclodextrins (β-CDs). The results showed that ultrasonic pretreatment (20 kHz and 38 W/L) at pH 6.0 promoted increased enzymatic activity. Specifically, after sonication at 25 °C/30 min, there was a maximum activity increase of 93 % and 68 % when biocatalysis was carried out at 25 and 55 °C, respectively. For activity measured at 80 °C, maximum increase (31 %) was observed after sonication at 25 °C/60 min. Comparatively, US pretreatment at low pH (pH = 4.0) resulted in a lower activity increase (max. 28 %). These activation levels were maintained after 24 h of storage at 8 °C, suggesting that changes on CGTase after ultrasonic pretreatment were not transitory. These pretreatments altered the conformational structure of CGTase, revealed by an up to 11 % increase in intrinsic fluorescence intensity, and resulted in macrostructural modifications, such as a decrease in particle size and polydispersion index (up to 85 % and 45.8 %, respectively). Therefore, the sonication of CGTase under specific conditions of pH, time, and temperature (especially at pH 6.0/ 30 min/ 25 °C) promotes macrostructural changes in CGTase that induce enzyme activation and, consequently, higher production of β-CDs.

A large-scale type I CBASS antiphage screen identifies the phage prohead protease as a key determinant of immune activation and evasion

Cyclic oligonucleotide-based signaling system (CBASS) is an antiviral system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by the recognition of viral DNA, but the molecular cues activating CBASS are incompletely understood. Using a screen of 975 type I CBASS operon-phage challenges, we show that operons with distinct cGAS/DncV-like nucleotidyltransferases (CD-NTases) and CD-NTase-associated protein (Cap) effectors exhibit marked patterns of phage restriction. We find that some type I CD-NTase enzymes require a C-terminal AGS-C immunoglobulin (Ig)-like fold domain for defense against select phages. Escaper phages evade CBASS via protein-coding mutations in virion assembly proteins, and acquired resistance is largely operon specific. We demonstrate that the phage Bas13 prohead protease interacts with the CD-NTase EcCdnD12 and can induce CBASS-dependent growth arrest in cells. Our results define phage virion assembly as a determinant of type I CBASS immune evasion and support viral protein recognition as a putative mechanism of cGAS-like enzyme activation.

Combined Effect of Gamma Radiation and Heavy Metals on Some Living Organisms.

The purpose of this study was to systematise scientific publications on the combined effect of gamma radiation and heavy metals on living organisms. For this purpose, the method of analysis was applied, by means of which scientific papers in PubMed, Google Scholar, and other related databases were analysed for compliance with the inclusion criteria, where the objects of research were toxic effects of radiation and heavy metals on cells and adaptation processes. The results revealed that the study of the problem was carried out on organisms such as microalgae, fungi, weed and agricultural plants, fish, laboratory rats, and human cell cultures. In most studies, an antagonistic effect between low doses of gamma radiation and heavy metal salts was reported, which was manifested by a reduction in the cytotoxicity of isolated exposure to each agent separately. However, there are studies showing additive effects, especially in heavy metals. At the molecular level, heavy metal accumulation in combination with low doses of radiation (typically defined as less than 0.1Gy or sievert) induces the expression of metallothionein proteins, which can bind free radicals. At the metabolic level, this is manifested by a decrease in lipid peroxidation products, activation of antioxidant enzymes, and a decrease in apoptosis. The study proved both a direct relationship between zinc and cadmium accumulation in cells and inhibition of caspases and an indirect one, by maintaining mitochondrial membrane integrity through metallothionein.

The ufmylation cascade controls COPII recruitment, anterograde transport, and sorting of nascent GPCRs at ER.

Ufmylation is implicated in multiple cellular processes, but little is known about its functions and regulation in protein trafficking. Here, we demonstrate that the genetic depletion of core components of the ufmylation cascade, including ubiquitin-fold modifier 1 (UFM1), UFM1 activation enzyme 5, UFM1-specific ligase 1 (UFL1), UFM1-specific protease 2, and UFM1-binding protein 1 (UFBP1) each markedly inhibits the endoplasmic reticulum (ER)-Golgi transport, surface delivery, and recruitment to COPII vesicles of a subset of G protein-coupled receptors (GPCRs) and UFBP1's function partially relies on UFM1 conjugation. We also show that UFBP1 and UFL1 interact with GPCRs and UFBP1 localizes at COPII vesicles coated with specific Sec24 isoforms. Furthermore, the UFBP1/UFL1-binding domain identified in the receptors effectively converts non-GPCR protein transport into the ufmylation-dependent pathway. Collectively, these data reveal important functions for the ufmylation system in GPCR recruitment to COPII vesicles, biosynthetic transport, and sorting at ER via UFBP1 ufmylation and interaction directly.

Enzyme-Activated Biomimetic Vesicles Confining Mineralization for Bone Maturation.

Inspired by the crucial role of matrix vesicles (MVs), a series of biomimetic vesicles (BVs) fabricated by calcium glycerophosphate (CaGP) modified polyurethane were designed to mediate the mineralization through in situ enzyme activation for bone therapy. In this study, alkaline phosphatase (ALP) was harbored in the porous BVs by adsorption (Ad-BVs) or entrapment (En-BVs). High encapsulation of ALP on En-BVs was effectively self-activating by calcium ions of CaGP-modified PU that specifically hydrolyzed the organophosphorus (CaGP) to inorganic phosphate, thus promoting the formation of the highly oriented bone-like apatite in vitro. Enzyme-catalyzed kinetics confirms the regulation of apatite crystallization by the synergistic action of self-activated ALP and the confined microcompartments of BVs. This leads to a supersaturated microenvironment, with the En-BVs group exhibiting inorganic phosphate (Pi) levels 4.19 times higher and Ca2+ levels 3.67 times higher than those of simulated body fluid (SBF). Of note, the En-BVs group exhibited excellent osteo-inducing differentiation of BMSCs in vitro and the highest maturity with reduced bone loss in rat femoral defect in vivo. This innovative strategy of biomimetic vesicles is expected to provide valuable insights into the enzyme-activated field of bone therapy.

Enzyme‐Assisted Activation Technique for Producing Versatile Hydrogel Microparticle Scaffolds with High Surface Chemical Reactivity

AbstractAdvancing the integration of nonliving and living components relies heavily on functional hydrogel materials with biocompatibility and customizability. In this study, an enzyme‐assisted surface activation method is developed to produce reactive hydrogel microparticles (HMPs) comprising thiolated hyaluronic acid and hyperbranched poly(β‐hydrazide esters). Fluorescence labeling analysis reveals an over six‐fold increase in surface‐active functional group density on the hydrogels and three‐fold on HMPs after enzyme activation. This enhancement improves accessibility of active elements, facilitating post‐functionalization and optimizing their capacity to support initial cell adhesion and spreading as carriers for cell cultures. Additionally, by utilizing the exposed reactive double bonds on the HMP surfaces post‐enzymatic treatment, thiolated HMPs are produced through a thiol‐ene coupling reaction with thiolated polymers. These thiolated HMPs bond together spontaneously, resulting in the formation of annealed granular hydrogels with interconnected large‐pore networks and a tunable storage modulus (G’) from tens to hundreds of pascals, compatible with most soft tissues. Integrated with 3D bioprinting, this hydrogel ink generates prints that foster cell adhesion, migration, growth, and network formation. Moving forward, integrating various granular hydrogel scaffold systems with the enzyme‐assisted activation technique holds significant promise for enhancing performance and expanding applications in regenerative medicine and innovative living materials.

Phosphoproteomic profiling of early rheumatoid arthritis synovium reveals active signalling pathways and differentiates inflammatory pathotypes

BackgroundKinases are intracellular signalling mediators and key to sustaining the inflammatory process in rheumatoid arthritis (RA). Oral inhibitors of Janus Kinase family (JAKs) are widely used in RA, while inhibitors of other kinase families e.g. phosphoinositide 3-kinase (PI3K) are under development. Most current biomarker platforms quantify mRNA/protein levels, but give no direct information on whether proteins are active/inactive. Phosphoproteome analysis has the potential to measure specific enzyme activation status at tissue level.MethodsWe validated the feasibility of phosphoproteome and total proteome analysis on 8 pre-treatment synovial biopsies from treatment-naive RA patients using label-free mass spectrometry, to identify active cell signalling pathways in synovial tissue which might explain failure to respond to RA therapeutics.ResultsDifferential expression analysis and functional enrichment revealed clear separation of phosphoproteome and proteome profiles between lymphoid and myeloid RA pathotypes. Abundance of specific phosphosites was associated with the degree of inflammatory state. The lymphoid pathotype was enriched with lymphoproliferative signalling phosphosites, including Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted novel kinases not previously linked to RA, such as Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotype. Several phosphosites correlated with clinical features, such as Disease-Activity-Score (DAS)-28, suggesting that phosphosite analysis has potential for identifying novel biomarkers at tissue-level of disease severity and prognosis.ConclusionsSpecific phosphoproteome/proteome signatures delineate RA pathotypes and may have clinical utility for stratifying patients for personalised medicine in RA.

From waste to strength: Tailor-made enzyme activation design transformation of denatured soy meal into high-performance all-biomass adhesive

Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance.

Light-independent pathway of STN7 kinase activation under low temperature stress in runner bean (Phaseolus coccineus L.)

BackgroundThe phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet.ResultsIn this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation.ConclusionsOur results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling.

Calcium enhanced the resistance against Phoma arachidicola by improving cell membrane stability and regulating reactive oxygen species metabolism in peanut

BackgroundPeanut (Arachis hypogaea), a vital oil and food crop globally, is susceptible to web blotch which is a significant foliar disease caused by Phoma arachidicola Marasas Pauer&Boerema leading to substantial yield losses in peanut production. Calcium treatment has been found to enhance plant resistance against pathogens.ResultsThis study investigates the impact of exogenous calcium on peanut resistance to web blotch and explores its mechanisms. Greenhouse experiments revealed that exogenous calcium treatment effectively enhanced resistance to peanut web blotch. Specifically, amino acid calcium and sugar alcohol calcium solutions demonstrated the best induced resistance effects, achieving reduction rates of 61.54% and 60% in Baisha1016, and 53.94% and 50% in Luhua11, respectively. All exogenous calcium treatments reduced malondialdehyde (MDA) and relative electrical conductivity (REC) levels in peanut leaves, mitigating pathogen-induced cell membrane damage. Exogenous calcium supplementation led to elevated hydrogen peroxide (H2O2) content and superoxide anion (O2∙-) production in peanut leaves, facilitating the accumulation of reactive oxygen species (ROS) crucial for plant defense responses. Amino acid calcium and sugar alcohol calcium treatments significantly boosted activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in peanut leaves. Activation of these antioxidant enzymes effectively scavenged excess ROS, maintaining ROS balance and mitigating cellular damage.ConclusionsIn summary, exogenous calcium treatment triggered ROS production, which was subsequently eliminated by the activation of antioxidant enzymes, thereby reducing cell membrane damage and inducing defense responses against peanut web blotch.

Detergent-resistant α-amylase derived from Anoxybacillus karvacharensis K1 and its production based on whey

In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg−1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg−1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg−1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5ʹ-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme’s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL−1 and 1580.3 ± 183.7 μmol mg−1 protein min−1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.