Enzymatic Parameters Research Articles

Seed and foliar application of nano-selenium improves sesame triacylglycerols and oil yield via photosynthetic pigment and enzymatic and chemical antioxidant enhancement revealed by spectrophotometric, UHPLC-analysis and chemometric modeling

The current study aimed to investigate the effects of plant-mediated selenium nanoparticles (SeNPs) on plant growth, photosynthetic pigments, antioxidant activity, and the triacylglycerol profile of sesame (Sesamum indicum L.). The green synthesis of SeNPs was achieved using garlic extract, resulting in spherical nanoparticles with an average size range of 70–75 nm. Three SeNP treatments (T3, 30 ppm; T4, 40 ppm; and T5, 50 ppm) were applied through seed and foliar spray on six sesame varieties (V1, TS-5; V2, TH-6; V3, Til-18; V4, Niab Millennium; V5, Niab Pearl; and V6, NS-16). All enzymatic antioxidant parameters showed an increase in the treated groups, such as SOD (74.4% in V1 at T4), POD (43% in V5 at T5), APX (62% in V1 at T3), and GPX (31.56% in V3 at T4). CAT showed the highest percentage improvement in T5 for V1, V2, V4, and V5, while V3 and V4 exhibited the highest values at T4. Likewise, seed antioxidant parameters also showed increase in antioxidant activity, highest total phenolic content (6.06 mg GAE/g) was found at T5 treatment with percent increase of 27.41%, but the highest percent increase was found to be at T4 treatments in V1 with increase of 46.83%. Percent oil yield was also noted to be higher as highest percent (60%) oil yield was obtained at T4 treatment in V3. Ultra High Performance Mass-Spectrometry (UHPLC-MS) analysis and chemometric modeling suggested a total of 10 triacylglycerol (TG) biomarkers separating untreated groups, with higher relative abundance values at T4 and T5 treatments compared to control. PCA and correlation analysis showed clustering of untreated groups from T4 and T5, which suggests that these two treatments result in higher accumulation of oil. A generalized linear model with ANOVA showed a highly significant impact of treatments on all the growth and oil parameters, with significance involvement of varieties. The interaction between variety and treatment showed no significant effect on the growth and oil biomarkers of sesame. However, it can be concluded that the T4 and T5 treatments (40 ppm and 50 ppm) of SeNPs, applied through seed and foliar methods, have a strong influence on the overall growth and oil yield of sesame. This warrants further transcriptomic and molecular analysis to gain deeper insight into the mechanisms of action of SeNPs.

Integrated Biomarker Response of Oxidative Stress Parameters in the Digestive Glands and Gills of Autochthonous and Invasive Freshwater Mussels from the Sava River, Serbia.

In this study, the activity of oxidative stress parameters superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST), as well as the concentrations of vitamin E (Vit E) and SH groups in the digestive glands and gills of freshwater mussels Unio pictorum and Sinanodonta woodiana from the Sava River in Serbia were investigated. These parameters were determined in native and invasive mussels under the same environmental conditions. The activities of GSH-Px and GR and the concentration of Vit E were significantly higher in the digestive glands of the autochthonous species U. pictorum than in the invasive species S. woodiana, while the CAT activity and the concentration of SH groups were lower. In the gills of U. pictorum, GSH-Px activity and Vit E concentration were significantly higher, while CAT, GST, and SH groups were lower. Principal component analysis (PCA) showed that oxidative stress parameters were strictly tissue- and species-specific. In addition, integrated biomarker response (IBR) showed a combined response of enzymatic and non-enzymatic oxidative stress parameters depending on the tissue or species studied, indicating different metabolic activities and behaviors of an autochthonous versus an introduced bivalve species.

Insight into physiological and biochemical markers against formaldehyde stress in spider plant (Chlorophytum comosum L.).

Formaldehyde is a prominent volatile organic compound and also considered as an indoor air pollutant. Chlorophytum comosum, an indoor plant, has been reported to metabolize indoor formaldehyde. But the phytotoxic effects of formaldehyde, being a pollutant, on C. comosum are not well explored. Furthermore, C. comosum responses that can be considered as markers at the physiological and biochemical level against formaldehyde stress are not yet investigated. Therefore, the current research study was aimed to evaluate such potential markers against formaldehyde in C. comosum. Briefly, C. comosum was exposed to 5-, 10-, and 20-ppm formaldehyde doses in an airtight glass chamber. Plant samples were then taken to analyze morpho-anatomical, physiological, and biochemical responses after short (2, 4, and 6h), medium (12 and 24h), and extended durations (48 and 96h) for each tested dose. Application of 10 and 20ppm formaldehyde doses leads to a significant incline in enzymatic antioxidants. Formaldehyde concentration of 10ppm leads to a maximum increase in catalase (30.30 U/mg of protein), guaiacol peroxidase (135.64 U/mg of protein), and superoxide dismutase (44.76 U/mg of protein) compared to their respective controls. A significant change is also observed in non-enzymatic parameters, including total phenolic content, which ranged from 3.62mg GAE/g (control) to 10.51mg GAE/g, total antioxidants vary from 27.37% (control) to 85.05% in 20ppm formaldehyde, respectively. However, formaldehyde application negatively affected the physiological responses of C. comosum by reducing its photosynthetic rate, transpiration rate, and stomatal conductance. Additionally, extended exposure of C. comosum to 10- and 20-ppm formaldehyde doses leads to visible leaf damage. Principal component analysis indicated that enzymatic parameters including SOD, CAT, and GPX and non-enzymatic parameters including MDA, TPC, TFC, TAOs, carotenoids, TSS, and intercellular CO2 contributed the most to the total variance. Thus, these parameters have potential to serve as physiological and biochemical markers in C. comosum against formaldehyde stress.

Lethal and sublethal concentrations spirodiclofen stress may increase the adaptation of Panonychus citri (Acari: Tetranychidae).

Panonychus citri is one of the most destructive pests in citrus orchards, exhibiting varying degrees of tolerance to numerous insecticides, such as spirodiclofen. To effectively manage pests, this study explores the response of P. citri to spirodiclofen stress from the perspectives of life history, enzymatic parameters, and reproduction. The effects of two concentrations (LC30 and LC50) of spirodiclofen on the biological parameters of P. citri were evaluated by the life table method. The results showed that the development duration, fecundity, oviposition days, and lifespan were shortened, though the pre-oviposition period of two treatments was prolonged in comparison with the control. A significant decrease was recorded in the net reproductive rate (R0) and the mean generation time (T) for the two treatments. Nevertheless, the intrinsic rate of increase (r) and the rate of increase (λ) were not significantly affected in the LC30 treatment, whereas they declined in the LC50 treatment. The enzyme activity assay resulted in higher activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and carboxylesterase (CarE), among the treatments than the control. In contrast, the treatments recorded lower cytochromeP450 (CYP450) and Glutathione S-transferase (GST) activities than the control. Furthermore, the study detected that relative mRNA expression of Vitellogenin (Vg) and Vitellogenin receptor (VgR) for two treatments were lower than the control. In summary, two concentrations of spirodiclofen inhibited progeny growth and fecundity of P. citri. Additionally, the results of this study may support further research on tolerance of P. citri in response to spirodiclofen stress.

Biochemical and kinetic properties of three indoleamine 2,3-dioxygenases of Aspergillus fumigatus: mechanism of increase in the apparent Km by ascorbate.

Indoleamine 2,3-dioxygenase (IDO) is a monomeric heme enzyme that catalyzes the oxidative cleavage of tryptophan (L-Trp) to form N-formyl-kynurenine. Similar to other heme proteins, IDO only binds to O2 when the heme iron is ferrous (FeII), thereby rendering the enzyme active. Thus, ascorbate (Asc, a reducing agent) and methylene blue (MB, an electron carrier) are commonly added to in vitro IDO assay systems. However, Asc and MB have been recently reported to significantly impact the measurement of the enzymatic parameters of vertebrate IDO. Aspergillus fumigatus is a filamentous fungus and the most common cause of invasive aspergillosis; it has three IDO genes (IDOα, IDOβ, and IDOγ). The FeII-O2 IDOs of A. fumigatus, particularly FeII-O2 IDOγ, have relatively long half-lives in their autoxidation; however, the autoxidation was accelerated by Asc. Similar to vertebrate IDOs, Asc acted as a competitive (or mixed-competitive) inhibitor of the IDOs of A. fumigatus. A positive correlation (in the order of IDOγ > IDOβ > IDOα) was observed between the inhibitory sensitivity of the IDOs to Asc and the facilitation of their autoxidation by Asc. The FeII-O2 IDO can repeat the dioxygenase reaction as long as it reacts with L-Trp; however, substrate-free FeII-O2 IDO is converted into inactive FeIII-IDO by autoxidation. Thus, L-Trp (which keeps the IDO active) competes with Asc (which inactivates IDO by accelerating autoxidation). This is probably why Asc, which is structurally quite different from L-Trp, appears to function as a competitive (or mixed-competitive) inhibitor of IDOs.

Investigation of Drought Stress on Chickpea (Cicer arietinum L.) Genotypes Employing Various Physiological Enzymatic and Non-Enzymatic Biochemical Parameters.

Drought stress is a universal crisis in sustaining the growth and production of major legumes, including the chickpea. Drought severely reduces the biomass of chickpea plants, with the effect on leaves appearing the most apparent. The aim of this study was to investigate, using various physiological and biochemical markers throughout the pod filling stage, how 78 desi chickpea genotypes tolerated drought stress. Most of the evaluated characteristics showed significant variations between control and drought treatments. The mean performance of most of the investigated parameters significantly decreased under moisture-stressed conditions. RWC, SWD, MSI, and CTD were investigated under terminal drought-stressed conditions. Except for saturated water deficit (SWD), all remaining characteristics declined with increasing stress. Genotypes SAGL152210, SAGL152252, SAGL152347, SAGL22-115, and JG11 were recognized as drought-tolerant based on physiological characteristics. Biochemical markers viz., protein content, total soluble sugar, lipid peroxidation, and proline content, had an impact on osmotic adjustment. Based on non-enzymatic biochemical traits, genotypes SAGL22-115, ICC4958, ICCV201108, ICCV201107, SAGL152252, and JG11 were identified for their capability to survive under drought-stressed conditions. H2O2 content, CAT, SOD, POD, APX, and DPPH were considered antioxidant agents. Genotypes SAGL152208, SAGL22-105, SAGL22-112, ICC201108, SAGL152278, SAGL152252, SAGL162371, SAGL162390, ICC 4958, and JG315 may be considered drought-tolerant based on antioxidant activities. These genotypes are believed to be better equipped with physio-biochemical mechanisms and antioxidant defense systems at the cellular level and can be used in breeding programs to breed drought-tolerant cultivar(s). They can also be screened in the future, allowing the line(s) that have remained consistent over time to be recognized and registered as drought-tolerant donors.

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax, and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

Wood biochar induced metal tolerance in Maize (Zea mays L.) plants under heavy metal stress

Due to metal toxicity, widespread industrialization has negatively impacted crop yield and soil quality. The current study was aimed to prepare and characterize biochar made from wood shavings of Pinus roxburghii and to determine the plant growth promoting and heavy metal detoxification of cadmium (Cd) and chromium (Cr) contaminated soil. FTIR SEM coupled with EDX characterization of biochar was performed; Cd and Cr were used at a rate of 20 mg/kg. Biochar was used at the rate of 50 mg/kg for various treatments. The completely randomized design (CRD) was used for the experiment and three replicates of each treatment were made. Various agronomic and enzymatic parameters were determined. The results indicated that all growth and enzymatic parameters were enhanced by the prepared biochar treatments. The most prominent results were observed in treatment T5 (in which shoot length, root length, peroxidase dismutase (POD), superoxide dismutase (SOD) catalyzes (CAT), and chlorophyll a and b increased by 28%, 23%, 40%, 41%, 42%, and 27%, respectively, compared to the control). This study demonstrated that biochar is a sustainable and cost-effective approach for the remediation of heavy metals, and plays a role in plant growth promotion. Farmers may benefit from the current findings, as prepared biochar is easier to deliver and more affordable than chemical fertilizers. Future research could clarify how to use biochar optimally, applying the minimum amount necessary while maximizing its benefits and increasing yield.

Characterization of a Xylosyltransferase from Panax notoginseng Catalyzing Ginsenoside 2'-O Glycosylation in the Biosynthesis of Notoginsenosides.

Notoginsenosides are important bioactive compounds from Panax notoginseng (Burk.) F. H. Chen, most of which have xylose in their sugar chains. However, the xylosyltransferases involved in the generation of notoginsenosides remain poorly understood, posing a bottleneck for further study of the biosynthesis of notoginsenosides. In this work, a new xylosyltransferase gene, PnUGT57 (named UGT94BW1), was identified from P. notoginseng, which has a distinct sequence and could catalyze the 2'-O glycosylation of ginsenosides Rh1 and Rg1 to produce notoginsenosides R2 and R1, respectively. We first characterized the optimal conditions for the PnUGT57 activity and its enzymatic kinetic parameters, and then, molecular docking and site-directed mutagenesis were performed to elucidate the catalytic mechanism of PnUGT57. Combined with the results of site-directed mutagenesis, Glu26, Ser266, Glu267, Trp347, Ser348, and Glu352 in PnUGT57 were identified as the key residues involved in 2'-O glycosylation of C-6 O-Glc, and PnUGT57R175A and PnUGT57G237A could significantly improve the catalytic activity of PnUGT57. These findings not only provide a new xylosyltransferase gene for augmenting the plant xylosyltransferase database but also identify the pivotal sites and catalytic mechanism of the enzyme, which would provide reference for the modification and application of xylosyltransferases in the future.

Optimization of the enzymatic clarification process of glucose syrups derived from agro-industrial residues

In this work, the application of pectinase enzymes to clarify glucose syrups produced from corn fiber was studied. The enzymatic activity and kinetic parameters of the pectinase enzyme used were quantified. A specific activity of 5,528 U/mg protein, a maximum rate of 19151 U/mL, and a Michaelis-Menten constant of 1,656 mg/mL were obtained. The syrup was prepared by hydrolyzing corn fiber at 50°C and 180 rpm, and a central composite design was performed for the clarification step to find the optimal conditions of enzyme-substrate ratio and agitation. The syrup was heated to a temperature of 50°C, and an enzyme substrate ratio between 2.5 to 4.5 U/mL of syrup and agitation, varying between 215 to 285 rpm, was applied. The conditions to maximize the clarification of corn fiber syrup were determined to be an enzyme substrate ratio of 3.716 U/mL syrup and agitation of 267 rpm. These conditions were validated by taking the syrup from clarity of 65.8% to 88.1%, demonstrating that the methodology used presents advantages in the syrup clarification process. A physicochemical characterization of the corn fiber used to prepare the syrups was carried out, which presented a content of 12.90%, 23.33%, 13.4%, and 0.36% of cellulose, hemicellulose, lignin and pectin, respectively.

MPEK: a multitask deep learning framework based on pretrained language models for enzymatic reaction kinetic parameters prediction.

Enzymatic reaction kinetics are central in analyzing enzymatic reaction mechanisms and target-enzyme optimization, and thus in biomanufacturing and other industries. The enzyme turnover number (kcat) and Michaelis constant (Km), key kinetic parameters for measuring enzyme catalytic efficiency, are crucial for analyzing enzymatic reaction mechanisms and the directed evolution of target enzymes. Experimental determination of kcat and Km is costly in terms of time, labor, and cost. To consider the intrinsic connection between kcat and Km and further improve the prediction performance, we propose a universal pretrained multitask deep learning model, MPEK, to predict these parameters simultaneously while considering pH, temperature, and organismal information. Through testing on the same kcat and Km test datasets, MPEK demonstrated superior prediction performance over the previous models. Specifically, MPEK achieved the Pearson coefficient of 0.808 for predicting kcat, improving ca. 14.6% and 7.6% compared to the DLKcat and UniKP models, and it achieved the Pearson coefficient of 0.777 for predicting Km, improving ca. 34.9% and 53.3% compared to the Kroll_model and UniKP models. More importantly, MPEK was able to reveal enzyme promiscuity and was sensitive to slight changes in the mutant enzyme sequence. In addition, in three case studies, it was shown that MPEK has the potential for assisted enzyme mining and directed evolution. To facilitate in silico evaluation of enzyme catalytic efficiency, we have established a web server implementing this model, which can be accessed at http://mathtc.nscc-tj.cn/mpek.

A review on textile solid waste management: Disposal and recycling.

Due to global population growth and living standards improvements, textile production and consumption are increased. Textile solid waste has become challenging issue for waste management authority. It is reported that textile materials are discarded daily, representing approximately 1.5% of the generated waste around the world. Over the past few decades, special attention has been given to the used clothes in all regions globally, which can reduce energy costs by 80% and also represent a source of raw materials economically profitable and environmentally responsible. This review article attempted to address different topics including: source of solid textile waste, environmental impact of textile waste as a result of massive consumption of clothing, textile waste management processes such as recycling, reuse of textile waste, landfill and incineration and energy recovery from textile waste. Narrative review with collection of recent quantitative information was carried to reflect the status of textile solid waste. In this article, the possibilities of bio-ethanol production from textile waste as valuable cellulosic raw material are investigated and presented. Results show that developing countries lack of systematic waste management. On another side of the globe, some countries are trying to recover energy these days by incineration. The heat and power that recovered from this process can be used instead of other energy sources. Throughout the incineration process, flue gases (CO2, H2O, O2, N2) are generated so it should be properly designed to avoid pollution. During energy recovery, different pre-treatment methods and different enzymatic hydrolysis parameters are recommended to be implied for better results.

Enhancing efficiency in ethanol production from high solid corn stover: Insights into enzymatic hydrolysis parameters and cellulase recovery

BackgroundThe simultaneous saccharification and co-fermentation (SSCF) at high solid loading is an effective approach to improve the economic feasibility of bioethanol. MethodsIn this study, the high-solid SSCF of superfine grinding corn stover was treated with cheap and readily available deionized water (DW) instead of sodium citrate buffer solution (SCBS). Additionally, the evaluation of temperature, enzyme loading, solid loading, and cellulose recovery were carried out to determine their impact on ethanol production. Significant FindingsThe results of the analysis indicated that replacing SCBS with DW had negligible effects on the SSCF of ethanol and byproducts (acetic acid and glycerol). The ethanol yield increased by 18 % at 35 °C compared to the yield at 30 °C. As enzyme loading increased, more cellulose active sites in corn stover were bound by cellulase, which increased the ethanol yield by improving the efficiency of enzymatic hydrolysis. The SSCF process didn't incorporate any buffer, resulting in an even higher ethanol titer (55.42 g/L). Furthermore, the cellulase productivity increased by 71 % after recovery, compared to the non-recovery. The results indicated that the optimized SSCF process is a potential technique for producing bioethanol on a commercial level because it improves the production efficiency while keeping costs low.

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation.

Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC-leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.

Enzymatic synthesis and parameters affecting on citronellyl acetate ester by trans-esterification reaction

Abstract Citronellyl acetate is a crucial component of flavor and fragrance in the food, cosmetic, and pharmaceutical sectors. In non-aqueous media, it can be successfully produced via lipase catalysis. This study focuses on the lipase-catalyzed trans-esterification of citronellol alcohol with geranyl acetate ester in a non-aqueous medium to produce citronellyl acetate. For the synthesis of citronellyl acetate, crude acetone powders isolated from several plant seedlings of black cumin, fenugreek, coriander, flax, and rape seed were examined for lipase activity. Black cumin seedling lipase had the highest level of citronella acetate production with a yield of 76.32% in 72 h of reaction time. To assess the impact of different reaction parameters on citronellyl acetate production in organic solvents, hexane was chosen as the best solvent, and black cumin seedling lipase was selected as the best biocatalyst. The highest conversion yield of ester (76.32%) was found when 0.25 M of geraniol acetate and 0.25 M of citronellol reacted at 41 oC after 72 h in the presence of 0.25 g of seedling lipase enzyme in n-hexane. It has been determined that crude black cumin seedling lipase is inexpensive yet effective and has the potential to be used industrially for the synthesis of terpene esters.

Biotic regulation of phoD-encoding gene bacteria on organic phosphorus mineralization in lacustrine sediments with distinct trophic levels

Organic phosphorus (Po) mineralization hydrolyzed by alkaline phosphatase (APase) can replenish bioavailable P load in the sediment water ecosystem of lakes. However, the understanding about the interaction between P load and bacteria community encoding APase generation in the sediment are still limited. Different P pools in the sediments from Taihu Lake, China were measured using sequential extraction procedure. The APAase activity (APA) were obtained accompanying with enzymatic dynamical parameters Vmax and Km. The abundances and diversity of gene phoD-harboring bacterial communities were assessed using high throughput sequencing. The analysis results showed the decrease of potentially bioavailable P fractions including MgCl2-P and Fe-P along sampling gradient southwards together with active P concentrations in the water. Conversely, increasing APA and absolute abundance of phoD gene were found with the decreasing of P loads southwards. Positive correlation (p < 0.05) between absolute abundance and APA indicated that phoD-encoding bacteria manipulated the APA and Po mineralization. Negative correlation (p < 0.01) suggested that the APA was restrained by high P load and was promoted under low P condition. However, higher Vmax and Km values suggested that high mineralization potential of Po maintained the high concentrations of potentially bioavailable P even the APA was restricted. The abundance increase of predominant genus Cobetia (from 15.51 to 24.34 %) mirrored by the reduced Calothrix abundance (from 24.65 to 1036 %) was speculated to be responsible for the APA promotion under low P condition. Higher diversity indices in the high P scenario suggested that high P load stimulated the ecological diversity of gene phoD-encoding bacteria community. Generally, rare taxa such as Burkholderia having high connected degrees in bacterial communities together with abundant genera synergistically manipulated the phoD gene abundance and APase generation. Interaction between P fractions and bacteria encoding phoD gene determined the eutrophication status in the lacustrine ecosystem.

Plant Growth Regulator from the Essential Oil of Syzygium aromaticum L. for Inhibition of Secondary Growth of Garlic Cultivated under Tropical Conditions

Garlic cultivation in tropical regions, such as the Brazilian Cerrado, faces the problem of secondary growth in the field induced by climatic conditions, which affects bulb quality and value. Clove essential oil (CEO) contains high levels of eugenol, which has the potential as an eco-friendly plant growth retardant (PGR) capable of reducing or inhibiting the secondary growth of bulbs in garlic cultivation. In this study, field experiments were carried out in two consecutive years (winter 2021 and 2022), spraying garlic plants with different concentrations of emulsion of CEO (0.0, 0.2, and 0.4%) in the differentiation phase; for comparison, the effects of water deficit, a prevalent agricultural technique in the region, were also evaluated. At a dose of 0.4%, the CEO reduced the prevalence of secondary growth and split bulbs without affecting yield. The mode of action of PGR was investigated by analyzing photosynthetic, enzymatic, and metabolomic parameters. The plants reduced amylolytic activity, and the photosynthetic parameters, after 7 days, were restored in both treatments. The analysis of the metabolomic profile of garlic leaves revealed changes in the pathways associated with the biosynthesis of fatty acids, wax, cutin, and suberin in plants treated with CEO, indicating possible damage to the surface coating of the leaf.

The influence of drug-induced metabolic enzyme activity inhibition and CYP3A4 gene polymorphism on aumolertinib metabolism

The purpose of this study is to clarify the drug interaction profile of aumolertinib, and the influence of CYP3A4 genetic polymorphism on aumolertinib metabolic characteristics. Through microsomal enzyme reactions, we screened 153 drugs and identified 15 that significantly inhibited the metabolism of aumolertinib. Among them, telmisartan and carvedilol exhibited potent inhibitory activities in rat liver microsomes (RLM) and human liver microsomes (HLM). In vivo, the pharmacokinetic parameters of aumolertinib, including AUC and Cmax, were significantly altered when co-administered with carvedilol, with a notable decrease in the clearance rate CLz/F. Interestingly, the pharmacokinetic parameters of the metabolite HAS-719 exhibited a similar trend as aumolertinib when co-administered. Mechanistically, both telmisartan and carvedilol exhibited a mixed-type inhibition on the metabolism of aumolertinib. Additionally, we used a baculovirus-insect cell expression system to prepare 24 recombinant CYP3A4 microsomes and obtained enzymatic kinetic parameters using aumolertinib as a substrate. Enzyme kinetic studies obtained the kinetic parameters of various CYP3A4 variant-mediated metabolism of aumolertinib. Based on the relative clearance rates, CYP3A4.4, 5, 7, 8, 9, 12, 13, 14, 17, 18, 19, 23, 24, 33, and 34 showed significantly lower clearance rates compared to the wild-type. Among the different CYP3A4 variants, the inhibitory potency of telmisartan and carvedilol on the metabolism of aumolertinib also varied. The IC50 values of telmisartan and carvedilol in CYP3A4.1 were 6.68 ± 1.76 μM and 0.60 ± 0.25 μM, respectively, whereas in CYP3A4.12, the IC50 exceeded 100 μM. Finally, we utilized adeno-associated virus to achieve liver-specific high expression of CYP3A4*1 and CYP3A4*12. In the group with high expression of the less active CYP3A4*12, the magnitude of the drug-drug interaction was significantly attenuated. In conclusion, CYP3A4 genetic polymorphism not only influences the pharmacokinetic characteristics of aumolertinib, but also the inhibitory potency of telmisartan and carvedilol on it.

Mechanistic Investigation of Enzyme Triggered Release from a Xyloglucan Matrix Tablet for Controlled Colonic Drug Delivery

The objective of this study was to investigate the mechanisms underlying drug release from a controlled colonic release (CCR) tablet formulation based on a xyloglucan polysaccharide matrix and identify the factors that control the rate of release for the purpose of fundamentally substantiating the concept and demonstrating its robustness for colonic drug delivery. Previous work demonstrated in vitro limited release of 5-aminosalicylic acid (5-ASA) and caffeine from these tablets in small intestinal environment and significant acceleration of release by xyloglucanase, an enzyme of the colonic microbiome. Targeted colonic drug delivery was verified in an animal study in vivo. In the present work, interaction of the xyloglucan matrix tablets with aqueous dissolution media containing xyloglucanase was found to lead to the spontaneous formation of a hydrated highly viscous gummy layer at the surface of the matrix which had a reduced drug content compared to the underlying regions and persisted with a nearly constant thickness that was inversely correlated to the enzyme concentration throughout the duration of the release process. Enzymatic hydrolysis of xyloglucan was determined to take place at the surface of the matrix leading to matrix erosion and a relation for the rate of enzymatic reaction as a function of bulk enzyme concentration and the concentration of dissolved xyloglucan in the gummy layer was derived. A mathematical model was developed encompassing aqueous medium ingress, matrix metamorphosis due to xyloglucan dissolution and matrix swelling, enzymatic hydrolysis of the polysaccharide and concomitant drug release due to matrix erosion and simultaneous drug diffusion. The model was fitted to data of reducing sugar equivalents in the medium reflecting matrix erosion and released drug amount. Enzymatic reaction parameters and reasonable values of medium ingress velocity, xyloglucan dissolution rate constant and drug diffusion coefficient were deduced that provided an adequate approximation of the data. Erosion was shown to be the overwhelmingly dominant drug release mechanism while the role of diffusion marginally increased at low enzyme concentration and high drug solubility. Changing enzyme concentration had a rather weak effect on matrix erosion and drug release rate as demonstrated by model simulations supported by experimental data, while xyloglucan dissolution was slow and had a stronger effect on the rate of the process. Therefore, reproducible colonic drug delivery not critically influenced by inter- and intra-individual variation of microbial enzyme activity may be projected.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.