Determination Of Enzyme Activity Research Articles

Crystal Structure and Mutagenesis of an XYP Subfamily Cyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity and Substrate Specificity.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of SsBcmA from Streptomyces sapporonensis. Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of SsBcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Analysis of Straw Degradation and Whole Genome of Acrophialophora multiforma.

Lignin is one of the main components in plants, which can transform value-added bioenergy and chemicals. At the same time, due to the close combination of lignin and hemicellulose in the structure, it becomes a barrier for cellulose utilization. Therefore, the effective degradation of lignin is of great significance for the utilization of these resources. In this study, the lignin degrading ability of Acrophialophora multiforma strain GZUIFR 22.397 was preliminarily investigated through straw degradation experiments and enzyme activity determination. Then, the whole genome of strain A. multiforma GZUIFR 22.397 was sequentially analyzed and annotated through multiple gene function annotation databases to comprehensively evaluate its lignin degrading potential. The results showed that the weight loss of straw reached 5.98 ± 3.95%. Laccase activity was 77.49 ± 2.65 U/L, lignin peroxidase activity was 160.57 ± 29.07 U/L, and manganese peroxidase activity was 294.83 ± 3.77 U/L. The genome of A. multiforma strain GZUIFR 22.397 spans 33.81 megabases and encompasses 9,370 genes. Among these, 6,122 genes have been annotated in the Gene Ontology (GO), 2,286 in the Cluster of Orthologous Groups of proteins (KOG), 2,283 in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and 603 in the Carbohydrate Active enZYmes Database (CAZy). Concurrently, the genome analysis predicted the presence of 9 genes for laccase, 2 genes for lignin peroxidase, and 2 genes for manganese peroxidase. In summary, these results indicated that A. multiforma GZUIFR 22.397 has lignin degrading ability, and laid the foundation for deciphering the molecular mechanism of A. multiforma GZUIFR 22.397 to degrade lignin.

PREDICTION OF THE STATE OF CRITICAL PATIENTS WITH SARS-CoV-2 INFECTION (LABORATORY ASPECT)

The aim of the study: to establish potential hematological markers for predicting mortality and recovery in patients with severe disease. Research metods. Demographic data, comorbidities, and blood parameters in patients with COVID-19 were analyzed. Critically ill COVID-19 patients have been divided into two research groups: those who recovered from a severe course of the disease (group 0) and deceased (group 1). Results and Conclusions. Clinical and laboratory hematocytological, biochemical, and hemostasis blood tests have been carried out. Diagnostic accuracy of several hematological indices has been established for prognostic stratification of the fatal outcome of the disease course in critical patients, namely the neutrophil-to-lymphocyte ratio (NLR > 5.57), the systemic immune- inflammation index (SII > 1914), enzyme activity determination (SPK > 137) and hemostasis parameters evaluation (D-dimer > 243; SFC > 6.5).

260 Industrial hemp (Cannabis sativa) supplementation improved plasma antioxidant enzymes and stress hormones in beef cattle

Abstract Industrial hemp is a rich source of antioxidants due to its high CBD but there is little information known about its impact on plasma antioxidant enzyme activity and stress hormone in beef cattle. The objective of this study was to determine the effect of industrial hemp supplementation on plasma antioxidant enzymes and stress hormone of Angus cattle. Black Angus heifers (n = 20) were completely randomized into either control (CON: n = 10, receiving 0 g of hemp) or hemp (HEMP: n = 10, receiving 30 g of hemp in 200 g of commercial concentrate) in a trial that lasted for 6 wk. Each group was replicated twice. Hay and water were offered ad libitum, experimental diets were offered o700 h daily. Blood samples were collected on d 0, 7, 14, 21, 28, 35 of the experiment for enzyme-linked immunosorbent assay to determine enzyme activity using standard kits. Data were analyzed using GLIMMIX procedure for repeated measure in SAS 9.4 with Compound symmetry as covariance structure while means were separated with Tukey at 5% alpha. There was significant (P < 0.05) difference in cortisol with HEMP group having 97.54 ng/mL and 122.23 ng/mL for CON. There was treatment x time interaction in total antioxidant capacity (TAOC) and malondialdehyde, TAOC range was 7.26 to 13.95 U/mL in HEMP group and 7.56 to 11.80 U/mL in CON. In conclusion, industrial hemp improved plasma antioxidant status, reduced lipid peroxidation and decreased stress level thereby improving the performance and welfare of beef cattle

The effects of long-term application of fomesafen on weed seedbank and resistance levels of Amaranthus retroflexus L.

Amaranthus retroflexus L. is one of the invasive malignant weeds in soybean fields. Diphenyl ether herbicides are commonly used to control weeds in soybean fields currently, among which fomesafen is the most widely used. With the long-term use of fomesafen, the weed species in soybean fields in multiple areas declined, and the control effect of fomesafen against Amaranthus retroflexus decreased significantly. This study aims to confirm the effects of long-term use of fomesafen on weed community richness and the current resistance level of Amaranthus retroflexus. In this paper, the result of seed germination indicated that the weed community richness decreased significantly due to the long-term application of fomesafen, and the primary dominant weed was Amaranthus retroflexus. The result of the whole-plant bioassay showed that Amaranthus retroflexus has developed resistance to fomesafen, and the resistance index was 50~59 g a.i. ha-1. The resistance level and mechanism of Amaranthus retroflexus were clarified by target gene detection, enzyme activity determination, and gene expression analysis. The results showed that there were both target resistance with Arg128Gly mutation in the PPX2 gene and non-target resistance (NTSR), with increased activity of metabolic enzymes (cytochromes P450 (P450s) and glutathione S-transferase (GSTs)) and protective enzymes (peroxidase (POD) and catalase (CAT)) in Amaranthus retroflexus.

Effects of Biochar-Amended Composts on Selected Enzyme Activities in Soils

This study examines the effect of biochar as an agricultural soil supplement on soil quality indicators, specifically enzyme activity in Missouri regions. While the benefits of biochar on soil bulk density, soil organic carbon, and infiltration have been established, its effect on soil enzyme activity has remained underexplored in this region. A three-year field investigation was conducted with six treatments (compost, biochar, compost + biochar, biochar + compost tea, fescue, and control) to evaluate the effects on enzymes such as β-glucosidase (BG), acid and alkaline phosphatases (ACP-ALP), arylsulfatase (ARS), dehydrogenases (DG), arylamidase (AMD), cellulase (CLS), and urease (URS). Furthermore, soil pH, organic matter (OM), and cation exchange capacity (CEC) were determined. The results showed that compost and biochar treatments considerably increased soil enzyme activity compared to other treatments, with nitrogen application further increasing enzyme activity. Soil pH, OM, and CEC were all important determinants in determining enzyme activity, with BG demonstrating strong positive associations with ACP and AMD (99.5%). This study shows that compost and biochar amendments significantly improve soil physicochemical and biological properties, thereby enhancing soil health and assisting farmers’ sustainable soil management practices.

Developmental exposure to arsenic reduces anxiety levels and leads to a depressive-like behavior in female offspring rats: Molecular changes in the prefrontal cortex

Exposure to inorganic arsenic (iAs) detrimentally affects the structure and function of the central nervous system. In-utero and postnatal exposure to iAs has been connected to adverse effects on cognitive development. Therefore, this investigation explores neurobehavioral and neurochemical effects of 0.05 and 0.10 mg/L iAs exposure during gestation and lactation periods on 90-day-old female offspring rats. The assessment of anxiety- and depressive-like behaviors was conducted through the application of an elevated plus maze and a forced swim test. The neurochemical changes were evaluated in the prefrontal cortex (PFC) through the determination of enzyme activities and α1 GABAA subunit expression levels. Our findings revealed a notable impact of iAs exposure on anxiety and the induction of depressive-like behavior in 90-day-old female offspring. Furthermore, the antioxidant status within the PFC exhibited discernible alterations in exposed rats. Notably, the activities of acetylcholinesterase and glutamate pyruvate transaminase demonstrated an increase, while glutamate oxaloacetate transaminase activity displayed a decrease within the PFC due to the iAs treatment. Additionally, a distinct downregulation in the mRNA expression of the α1GABAA receptor was observed in this neuronal region. These findings strongly suggest that iAs exposure during early stages of rat development causes significant modifications in brain oxidative stress markers and perturbs the activity of enzymes associated with cholinergic and glutamatergic systems. In parallel, it elicits a discernible reduction in the level of GABA receptors within the PFC. These molecular alterations may play a role in the diminished anxiety levels and the depressive-like behavior outlined in the current investigation.

Effect of supplemental milk replacer and liquid starter diet for 4 and 11 days postweaning on intestinal parameters of weaned piglets and growth to slaughter

Reduced piglet feed intake immediately postweaning (pw) leads to disruption of small intestine structure and function and reduced growth. Our objective was to evaluate the effect of providing supplemental milk or liquid starter diet for either 4 or 11 days pw, on intestinal parameters of newly weaned piglets and growth to slaughter. At weaning (28 ± 0.6 days old), five hundred and eighty−seven piglets ((Large White × Landrace) × Duroc) were divided into 59 pen groups, each containing 9–10 same sex (entire male or female) piglets. The pen groups were blocked by sex and weaning weight and provided with ad-libitum access to one of five dietary treatments: (1) Dry pelleted starter diet (control; CON); (2) CON+liquid milk replacer for 4 days pw (M4); (3) CON+liquid milk replacer for 11 days pw (M11); (4) CON+liquid starter diet for 4 days pw (S4) and (5) CON+liquid starter diet for 11 days pw (S11). Pen groups were weighed at weaning, days 11, 20, 28, and 47 pw and at target sale weight. Feed disappearance (on a DM basis) was recorded on each weighing day. On day 7 pw, 10 piglets per treatment were euthanised to collect small intestine tissue samples for determination of villus height (VH), crypt depth and brush-border membrane enzyme activity. Data were analysed using SAS-version 9.4. Between days 0 and 11 pw, M11 increased average daily feed intake by 48% and average daily gain (ADG) by 57% compared to CON (P < 0.05), and increased ADG by 54% (P < 0.05) compared to S4. Piglets on M11 also had improved feed conversion efficiency compared with CON piglets between days 0 and 11 pw. Treatment did not affect growth performance after day 28 pw, or carcass parameters at slaughter. At day 7 pw, M11 piglets had 37% higher jejunal VH than CON piglets (P < 0.05) and S11 piglets had 28% higher ileal VH than S4 piglets (P < 0.05). M11 piglets had up to 150% higher ileal sucrase activity than M4, S4 and S11 piglets (P < 0.05) and 180% higher ileal maltase activity than S4 piglets (P < 0.05). In conclusion, M11 reduced the immediate negative effects of weaning, as it was associated with increased feed intake, growth, brush-border membrane enzyme activity and improved intestinal structure early pw. However, there were no carryover effects of any of the liquid supplements on growth or feed efficiency or carcass weight at slaughter.

Through virtual saturation mutagenesis and rational design for superior substrate conversion in engineered d-amino acid oxidase.

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.

The Effect of Neuronal CoQ10 Deficiency and Mitochondrial Dysfunction on a Rotenone-Induced Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder currently affecting the ageing population. Although the aetiology of PD has yet to be fully elucidated, environmental factors such as exposure to the naturally occurring neurotoxin rotenone has been associated with an increased risk of developing PD. Rotenone inhibits mitochondrial respiratory chain (MRC) complex I activity as well as induces dopaminergic neuronal death. The aim of the present study was to investigate the underlying mechanisms of rotenone-induced mitochondrial dysfunction and oxidative stress in an in vitro SH-SY5Y neuronal cell model of PD and to assess the ability of pre-treatment with Coenzyme Q10 (CoQ10) to ameliorate oxidative stress in this model. Spectrophotometric determination of the mitochondrial enzyme activities and fluorescence probe studies of reactive oxygen species (ROS) production was assessed. Significant inhibition of MRC complex I and II-III activities was observed, together with a significant loss of neuronal viability, CoQ10 status, and ATP synthesis. Additionally, significant increases were observed in intracellular and mitochondrial ROS production. Remarkably, CoQ10 supplementation was found to reduce ROS formation. These results have indicated mitochondrial dysfunction and increased oxidative stress in a rotenone-induced neuronal cell model of PD that was ameliorated by CoQ10 supplementation.

Quantitative analyses of major enzyme activities in postharvest fruit

AbstractThe quantitative determination of major enzyme activities in postharvest fruit is a necessary biochemical experiment to investigate the physiological and metabolic mechanisms of fruit ripeness, senescence, and stress responses. However, it is necessary to choose appropriate methods to stably extract and determine target analytes from complex plant matrices due to the dynamic changes in enzyme activities of postharvest fruit during storage. Here, we outline sensitive and accurate methods for measuring fruit enzyme activities associated with key physiological processes including respiration, reactive oxygen species scavenging, membrane lipid degradation, energy synthesis, cell walls degradation, and disease resistance. This manuscript aims to provide guidance for future research in postharvest biology.

THE GLA D313Y MUTATION IN FABRY DISEASE: DIAGNOSTIC DILEMMAS AND THERAPEUTIC CONSIDERATIONS

Abstract Fabry disease is a genetic disorder caused by mutations in the GLA gene, which encodes alpha–galactosidase A, leading to enzyme dysfunction and subsequent accumulation of glycosphingolipids. In the classical form, it occurs in childhood or early adolescence with renal, cardiac, and neurological involvement, resulting in a poor prognosis. The GLA D313Y is a missense mutation resulting in a significant reduction of enzyme activity in plasma, while leukocyte enzyme activity remains within normal limits, leading to a "pseudo–deficiency" of enzyme activity with uncertain pathogenicity. In literature, it has been observed that individuals with this mutation may present predominantly with central neurologic manifestations, characterized by multifocal white matter lesions. Historically they do not receive indications for initiating enzyme replacement or molecular chaperone therapies. We present the case of a man affected by arterial hypertension and history of chronic lower limb pain who experienced a lacunar stroke at the age of 35. The GLA D313Y mutation was identified and enzyme activity assay in plasma was within normal limits, precluding the initiation of enzyme replacement therapy. Over the following years, he suffered two additional ischemic strokes, a progressive decline in renal function to stage 3, and acute coronary syndrome. Transthoracic echocardiography revealed hypertrophic non–obstructive cardiomyopathy (SIVd 20 mm) with normal biventricular ejection fraction. Cardiac magnetic resonance showed late gadolinium enhancement of inferior junctional and inferior wall in middle segment (Fig.1). Considering the patient‘s clinical conditions involving multi–organ impairment, an enzymatic activity test was performed on leukocytes, revealing a reduced activity. This prompted the decision to initiate enzyme replacement therapy. This case report supports the hypothesis that the D313Y mutation may be associated with a later presentation and not only neurological involvement. In our experience, it’s mandatory to proceed with the determination of leukocyte enzyme activity in presence of normal enzyme activity in plasma. As an alternative, the detection of glycosphingolipids’ accumulation could be useful, but myocardial and renal biopsy poses a high bleeding risk, especially in anticoagulated or antiaggregated patients. The authors propose considering the initiation of therapy in patients with the D313Y mutation who exhibit significant organ involvement.

Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and ∼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 μM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 μM) and mGPDH (Ki 54.70 μM) activities. siRNAs transfection showed ∼50 % and ∼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Effect of the Oligosaccharid Fraction of Fusarium oxysporum f. sp. vasinfectum on the Activity of Phenolic Metabolism Enzymes in Cotton Leaves

The synthesis of secondary metabolites, notably phenolic compounds, is part of the plant's defense reactions. The enzymes of phenolic metabolism can constitute a parameter of measurement of the defense of the plants. The aim of this work is to evaluate the activity of phenolic metabolism enzymes and an antioxidant enzyme after treatment of cotton plants with a fungal elicitor. Cotton plants obtained from the cultivar Y764AG3 were treated with the oligosaccharide fraction (FOS) of Fusarium oxysporum f. sp. Vasinfectum (FOV). The extraction of the enzymes is carried out cold by grinding the leaves in extraction buffer in the presence of PVP (0.5 %) and 0.1 M sodium phosphate buffer. The supernatant obtained after centrifugation of the filtrate was purified with Dowex-2 in order to obtain the purified enzymatic fraction. This fraction was used for the determination of enzyme activity by reading the absorbances. The results showed that the activity of ammonia-lyase activity was higher in the treated plants than in the control. This reflects an increase in the biosynthesis of phenolic compounds in the treated plants. On the other hand, oxidoreductase activity was higher in control plants than in treated plants. This indicates a decrease in the degradation of phenolic compounds in the treated plants. This study revealed the existence of an active phenolic metabolism in the FOS-treated plants. Thus defence mechanisms are activated in plants treated with the oligosaccharide fraction of Fusarium oxysporum f. sp. Vasinfectum.

N-succinyl-chitosan as ecofriendly pesticide carriers: Nano encapsulation and synergistic antifungal effect on 4-hydroxyphenyl-2-propenyl-1-one derivatives based on chalcone structure

IntroductionTraditional pesticides have poor-water solubility, high toxicity and low bioavailability. Therefore, it is of great significance for the sustainable and healthy development of the pesticide industry to develop efficient and ecofriendly new chemical pesticide products and formulations. ObjectivesThis study aims to synthesize a series of derivatives based on chalcone structure (HPPO), and then use the amphiphilic and self-assembly characteristics of N-succinyl-chitosan (NSCS) to prepare HPPO@NSCS nanoparticles (HPPO@NSCS NPs) in order to realize the green application of HPPO, and investigate the antifungal activity and mechanisms of HPPO@NSCS NPs. MethodsNSCS was synthesized by structural modification using chitosan as the carrier. Based on its amphiphilic and self-assembly characteristics, HPPO-16@NSCS NPs were reasonably prepared by combining with active small molecule HPPO-16. Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), fluorescence spectroscopy (FS) and high-performance liquid chromatography (HPLC) were used to characterize the physicochemical properties of NSCS and HPPO-16@NSCS NPs. The inhibitory activity of nanopesticides against Rhizoctonia solani (R. solani) was tested in vivo and in vitro. The mechanism of antifungal action was discussed from the observation of pathogen morphology, fluorescence staining and enzyme activity determination. Results28 small molecules based on chalcone structure (HPPO-1-28), NSCS and HPPO-16@NSCS were successfully synthesized. The application of HPPO-16@NSCS could impair the development, cell structure, cellular energy utilization, and metabolism pathways of the fungi. The protective effects of HPPO-16@NSCS NPs on rice leaves and leaf sheaths were 80.9 and 76.1 %, respectively, which were better than those of azoxystrobin. ConclusionThis study reveals that these simple chalcone derivatives can be further explored as viable antibacterial alternatives and NSCS as a novel pesticide matrix can be used for the delivery of more insoluble pesticides.

Changes in growth, morphology, and levels of digestive enzymes and growth-related hormones in early ontogeny of black scraper, Thamnaconus modestus

IntroductionThe black scraper, Thamnaconus modestus, is a highly valued marine fish species, but its output has gradually decreased in recent years, which may be due to its low survival rate during early ontogenesis.MethodsTherefore, in this study, we assessed the changes in growth, morphology, digestive enzymes, and hormone levels in T. modestus from 0–60 days post-hatching (dph) and revealed growth turning points by morphological measurement and determination of digestive enzyme activities and hormone levels. We found that ontogenesis could be divided into the larval (0–20 dph) and juvenile (20–60 dph) stages. Acid and alkaline protease activity significantly increased and decreased, respectively, from 12–25 dph, likely due to the development of stomach and gastric glands. Acid phosphatase levels significantly increased at 0 and 4 dph, which may be related to the regulation of metabolism and immune protection. A sharp increase in alkaline phosphatase levels at 20 and 25 dph was observed and was likely due to the development of the brush border membrane of enterocytes. The amylase level was significantly higher at 25, 30, and 35 dph, possibly due to better digestion and absorption during the transition from consuming Artemia to compound feed. In newly hatched larvae, the level of thyroid hormones triiodothyronine (T3) and thyroxine (T4) gradually increased and peaked at 35 dph, highlighting the importance of these hormones during the development of T. modestus. Growth hormone (GH) levels first increased from 0–8 dph, with a plateau at 8–20 dph, and then increased at 25–30–35 dph. For insulin-like growth factor 1 (IGF-1), a significant increase with a subsequent plateau was observed between 8 and 20 dph, followed by a substantial decrease between 30 and 35 dph. These results suggest that the regulating functions of GH and IGF-1 are synchronised. Digestive enzyme activity and hormone levels of abnormal fry at 30 dph were lower than those of normal fish, highlighting the importance of specific hormones, especially T4 and IGF-1, in the development of T. modestus.

Glucose Catabolite Repression Participates in the Regulation of Sialidase Biosynthesis by Antarctic Strain Penicillium griseofulvum P29.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

WITHDRAWN: Isolation and Functional Study of Efficient Cellulose Degrading Bacteria from Horse Cecum Contents

ObjectivesAnimals that consume and digest roughage for a long time have unique gut microbiota in their gastrointestinal tract. Studying the isolation of beneficial functional bacteria from the gut can provide resources for the development of feed microbial additives. Therefore, the purpose of this study was to isolate and identify bacteria with cellulose degradation ability from the cecal contents of Yili horses, and to provide resources for the development of feed microbial additives. MethodsFirstly, the cecal contents of 18 Yili horses were collected, and cellulose degrading bacteria were isolated and screened using CMC-Na screening medium under aerobic and anaerobic conditions. The strains with strong cellulose degradation ability were identified using Congo red staining and DNS methods. The top 60 strains with enzyme index were selected, and full-length 16S rRNA sequencing technology was used to identify and construct a phylogenetic tree. Select the top 5 strains of bacteria with high enzyme activity based on the measured cellulase activity, and the sheep were treated with mixed bacterial suspension by gavage. The rumen microbiota of sheep was analyzed using 16S rDNA amplicon sequencing technology. ResultsA total of 263 single colonies were isolated, of which 120 single colonies had a hydrolysis circle and colony enzymatic index (EI) of >1.5. Enzyme activity determination screening of the top 60 strains of full-length 16S rRNA sequencing, sequencing results show that the strains are mostly Bacillus spp. Among the 60 strains of bacteria, CE207 has the highest enzyme activity of 14.81 U/mL, and the lowest enzyme activity of CE105 is 1.91 U/mL. The mixed bacteria significantly increased the daily weight gain of sheep (P < 0.001) and feed intake (P < 0.001), and feed conversion rate increased from 7.75% to 10.09%. Sequencing results of sheep rumen microorganisms showed that, compared with the control group, the experimental group contained Muribaculum, Parvibacter, Desulfovibrio, Novosphingobium, Cutibacterium, Parasutterella, Lachnospiraceae_UCG-006, and Allobaculum genera increased significantly in abundance, and Fibrobacter and NK4A214_group genera decreased significantly in abundance, suggesting that a mixture of bacteria can influence the changes in the microflora of the rumen intestinal tract of sheep. ConclusionsThis finding suggests that the cellulose degrading bacteria isolated in the study can effectively promote the growth and development of herbivores such as sheep, and that efficient cellulose degrading bacteria in the animal gut may become a new direction for the development of feed additives in the future.

Integrated metabolic phenotypes and gene expression profiles reveal enzymatic browning mechanism in postharvest chrysanthemum

Chrysanthemum (Chrysanthemum morifolium Ramat) is a valuable crop mainly used for medicine, beverages and ornament. Enzymatic browning causes serious postharvest flower deterioration of chrysanthemum, but the mechanism remains unclear. To clarify the mechanism involved, the cellular ultrastructure, metabolites, browning products, and differentially expressed genes of chrysanthemum were studied systematically. During the browning process, cellular compartmentalization was severely disrupted and the contents of chlorogenic acid derivatives and flavonoid glycosides decreased significantly. A browning product with a dihydrobenzofuran skeleton was purified and identified for the first time from a simulated browning reaction of chlorogenic acid catalyzed by peroxidase. Moreover, six differentially expressed genes were for the first time identified to be involved in the regulation of oxidase by transcriptome and qPCR, and one of which (c76073_g1) was validated using in vitro enzyme activity determination. This research elucidates the inherent mechanism of enzymatic browning, provides a scientific explanation for the steam-treated processing of chrysanthemums and lays the foundation for the molecular breeding of anti-browning chrysanthemum varieties.

Non-canonical amino acids uncover the significant impact of Tyr671 on Taq DNA polymerase catalytic activity.

Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus. Five non-canonical amino acids, namely l-3,4-dihydroxyphenylalanine (l-DOPA), p-aminophenylalanine (pAF), p-acetylphenylalanine (pAcF), p-cyanophenylalanine (pCNF) and p-nitrophenylalanine (pNTF), were individually incorporated at position 671. Strikingly, Y671pAF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671pAF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probethe role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.