Screening system for MAA-CoA productivity using 2-methylcitrate biosensor.

A study was undertaken to estimate the activities of the key enzymes of glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle in purified rat spermatocytes and spermatids, which have been shown to die in glucose-containing medium and require lactate/pyruvate for maintaining normal ATP concentrations. The aim was to elucidate the changes in the glycolytic and oxidative potential of germ cells undergoing meiosis. Pachytene spermatocytes and round spermatids from adult rat testis were purified to approximately 90% purity by trypsin digestion followed by a combination of centrifugal elutriation and Percoll density gradient centrifugation. After the purity and viability of these cells had been established, their contents of hexokinase, phosphofructokinase, lactate dehydrogenase (LDH) and LDH-X of glycolysis, glucose 6-phosphate dehydrogenase of the pentose phosphate pathway and citrate synthase, aconitase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of the TCA cycle were estimated. These enzymes were also estimated in epididymal spermatozoa for comparison with the testicular germ cells. The results indicate greater activity of glycolytic and pentose phosphate pathway enzymes in spermatocytes than in spermatids, which exhibited greater activity of TCA cycle enzymes than the former. The difference in activity was statistically significant for most of the enzymes studied. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes and significantly lower activity of pentose phosphate pathway and TCA cycle enzymes than did the testicular germ cells. We conclude that the unusual dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilise lactate, pyruvate, or both, while retaining a better ability to utilise glucose. Spermatozoa with the greatest glycolytic potential and the lowest TCA cycle activity appear to be 'programmed' to utilise exclusively glucose/fructose for energy.

Ergot alkaloids are mycotoxins that negatively affect humans and animals but also have useful pharmaceutical properties. Ergot alkaloids have been extensively studied in Claviceps purpurea, and also have been reported in other fungi, such as certain Penicillium spp., Aspergillus fumigatus, and several Neotyphodium spp. that are endophytes of grasses. There is a high degree of variation in the production of ergot alkaloids within an individual species and among producers from different taxa, which means different fungi accumulate different profiles of ergot alkaloids rather than a single pathway end product. These varied profiles result, in part, from inefficiency in the pathway which leads to increased accumulation of certain pathway intermediates in addition to the end product of the pathway. These intermediates have different biological activities and provide different benefits to the host than the pathway end products. I conducted a series of experiments to investigate the potential mechanisms by which fungi accumulate different profiles of intermediates and end products. In A. fumigatus, efficiency in completing the pathway varied developmentally. Wild type conidiating cultures containing all cell types involved in conidiation (hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated more of the pathway intermediate festuclavine and less of the pathway end product fumigaclavine C than did isolated mature conidia. Such differences also were indicated by studies with a medA mutant, which proliferates phialides. The medA mutant accumulated more pathway intermediates than did the wild type or a complemented medA mutant. A stuA mutant, lacking recognizable conidiophores, produced no detectable ergot alkaloids. Although this variation in alkaloid accumulation indicated a difference in distribution or activity of ergot pathway enzymes, fluorescence associated with the expression of five tested alkaloid pathway genes indicated a similar pattern of expression of each gene in the different cell types. Fluorescence was not observed in young hyphae but was detected at the apices of hyphae as they swelled to form the vesicle of the conidiophore. More fluorescence was observed in mature vesicles, phialides, and conidia. No qualitative differences were observed in cell type-specific expression of different pathway genes, indicating that observed differences in accumulation of intermediates versus end product must be due to quantitative differences in expression or differences in activity of pathway enzymes. In Neotyphodium lolii x Epichloe typhina isolate Lp1, an endophytic symbiont of perennial ryegrass, different ergot alkaloid profiles were observed in different tissues of the host plant. RNA interference (RNAi) and overexpression of

Bioderived nanostructure and their functional contribution in immobilization of cellulolytic enzymes for improving biosensing application

Enzyme-catalyzed CO2 reduction to value-added commodities is important for alleviating the global environmental issues and energy crises due to high selectivity and mild conditions. Owing to high energy density, formic acid or methanol produced from CO2 using formate dehydrogenase (FDH) or multi-enzyme cascades are promising target chemicals for CO2 utilization. However, the low activity, poor stability and low reusability of key enzymes involved in such process hampered its large-scale application. Enzyme immobilization provides an effective solution to these problems and significant progress have been made in immobilization carriers. Moreover, integration of enzyme immobilization with other catalysis techniques have been explored extensively. This review summarized the recent advances in the immobilization of enzymes using membranes, inorganic materials, metal-organic frameworks, covalent organic frameworks and other carriers, and illustrated the characteristics and advantages of different immobilization materials and immobilization methods. The synergistic effects and applications of immobilized enzymes and electrocatalytic or photocatalytic coupling reaction systems for CO2 reduction were further summarized. Finally, the current challenges of enzyme immobilization technology and coupling reaction systems were pointed out and their development prospects were presented.

To clarify the regulatory mechanism underlying the interaction between air relative humidity (RH) and soil moisture (field capacity, FC) on sugar accumulation in tomato fruits, this study used two tomato cultivars, 'Xiuzhen' and 'Jinhong 208', as materials. Four RH levels (40%, 60%, 80%, and 95%) and two FC levels (40% and 80% FC) were established in artificial climate chambers to systematically analyze the relationships among photosynthesis, transpiration, vascular transport, sugar metabolism enzyme activity, and sugar accumulation. The results showed that low soil moisture (40% FC) reduced the photosynthetic rate but significantly increased sugar metabolism enzyme activity, thereby promoting sugar accumulation. In contrast, high soil moisture (80% FC) was associated with higher photosynthetic rates but lower sugar-metabolizing enzyme activity. High air humidity (95% RH) markedly associated with lower photosynthesis, stomatal conductance, and sugar metabolism enzyme activity, accompanied by a significant decrease (P < 0.01) in glucose, fructose, and sucrose contents; conversely, low humidity promoted sugar accumulation. Both 'Xiuzhen' and 'Jinhong 208' achieved the highest soluble sugar content under 40% RH and 40% FC conditions. Gray relational analysis revealed that FC had a stronger influence on photosynthesis, while RH had a more pronounced effect on sugar metabolism. Under sufficient water supply, air humidity management played a greater role in regulating fruit quality. Path analysis indicated that RH exerted significant negative effects on sugar accumulation via reduced transpiration (path coefficient = -0.590), sugar metabolism enzyme activity (-0.358), and phloem transport (-0.424). In contrast, FC promoted phloem transport (0.680) but somewhat associated with lower enzyme activity (-0.500). Humidity is closely related to sugar accumulation, but weakly related to yield and its components. This study reveals the specific strategies of sugar regulation among different tomato cultivars and provides insights for humidity and irrigation optimization in greenhouse tomato production.

Deficiency of beta-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced beta-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low beta-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the beta-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and 35SO4-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low beta-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low beta-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low beta-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801deltaT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold.

Exocrine pancreatic insufficiency (EPI) reflects inadequate pancreatic enzyme(s) and/or bicarbonate secretion, leading to malabsorption. We aim to compare different assessment tools for exocrine pancreatic function and correlate them with the endoscopic pancreatic function test (ePFT) as a reference standard. Combined retrospective and prospective analysis of clinical data for children who underwent clinically indicated ePFTs. We compared ePFT against patient symptoms, laboratory results, magnetic resonance cholangiopancreatography with secretin 49/53 magnetic resonance imaging, among those, 35/49 with secretin enhanced magnetic resonance cholangiopancreatography(sMRCP), or endoscopic ultrasound (EUS 36/53) findings with the ePFT results. A total of 53 patients who underwent ePFT between September 2018 and April 2023 were included. 30 were Female (57%). Median age when ePFT was performed was 14 years (range: 4-21 years). Primary indication for ePFT: Acute recurrent or chronic pancreatitis (74%), Pancreas atrophy (9%), Other (post- pancreatic surgery, malnutrition with low fecal elastase-1 (FE-1) (17%). Patients with highpancreatic enzyme activities had a higher volume of pancreatic fluid secretion on ePFT. They also had larger pancreatic anteroposterior diameter and pancreas parenchyma volume, as well as higher post-secretin enteric fluid volume increment on sMRCP compared to those with low enzyme activities. Our findings also underscore the importance of lipase in maintaining and promoting weight gain in pediatric patients. In our patient cohort, the FE-1 level was lower in patients with low pancreatic enzyme activities. Our study showed that steatorrhea is associated with low pancreatic enzyme activities, whereas abdominal pain and unintentional weight loss might be nonspecific to EPI. No significant correlations were observed from blood cell counts, blood electrolytes, lipase, albumin, bilirubin, alanine aminotransferase, serum lipids, fat-soluble vitamins, prothrombin time, hemoglobin A1C, and EUS with pancreatic enzymes or bicarbonate concentration. sMRCP is a promising noninvasive modality in assessing exocrine pancreatic function in children.

Effect of ionic liquid on activity, stability, and structure of enzymes: A review

In this dispensation of the fourth industrial revolution, protein engineering has become a popular approach for increasing enzymatic activity, stability, and titer in the biosynthesis of natural products. This is attributed to its numerous advantages (over direct isolation from plants or via chemical synthesis), including decreasing or eliminating reaction byproducts, high precision, moderate handling of intricate and chemically unstable chemicals, overall reusability, and cost efficiency. Recently, protein engineering tools have advanced to redesign and enhance natural product biosynthesis. These methods include direct evolution, substrate engineering, medium engineering, enzyme engineering and immobilization, structure-assisted protein engineering, and advanced computational. Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article. Also, the advantages, limitations, and applications in industrial and medical biotechnology were discussed. Last, future research directions and potential were also highlighted.

Purine metabolism in promyelocytic HL60 and dimethylsulphoxide-differentiated HL60 cells

Natural laccase is an environmentally friendly biocatalyst in the degradation of a broad range of toxic pollutants because its catalysis reaction does not require or produce toxic reactants and byproducts. However, its inherent limitations, such as operational sensitivity, poor stability, and difficulty in recovery/reusability, have significantly restricted its practical environmental applications. Consequently, in recent years, researchers have focused on the development of sustainable catalysts to mimic natural laccase. This review focuses on biomolecule-based laccase mimics, which are derived from nucleotides, nucleic acids, amino acids, peptides, and proteins, summarizing their environmental applications. These biomolecule-based laccase mimics not only overcome the limitations of natural laccase by offering advantages such as high stability, ease of recycling, and long-term storage but also exhibit excellent biodegradability, making them green and sustainable catalytic materials. This study aims to present recent progress in biomolecule-based laccase mimics, as well as their challenges, and to offer future directions in laccase-like catalysts for environmental applications.

Computerized aiding systems can assist human decision makers in complex tasks but can impair performance when they provide incorrect advice that humans erroneously follow, a phenomenon known as “automation bias.” The extent to which people exhibit automation bias varies significantly and may reflect inter-individual variation in the capacity of working memory and the efficiency of executive function, both of which are highly heritable and under dopaminergic and noradrenergic control in prefrontal cortex. The dopamine beta hydroxylase (DBH) gene is thought to regulate the differential availability of dopamine and norepinephrine in prefrontal cortex. We therefore examined decision-making performance under imperfect computer aiding in 100 participants performing a simulated command and control task. Based on two single nucleotide polymorphism (SNPs) of the DBH gene, −1041 C/T (rs1611115) and 444 G/A (rs1108580), participants were divided into groups of low and high DBH enzyme activity, where low enzyme activity is associated with greater dopamine relative to norepinephrine levels in cortex. Compared to those in the high DBH enzyme activity group, individuals in the low DBH enzyme activity group were more accurate and speedier in their decisions when incorrect advice was given and verified automation recommendations more frequently. These results indicate that a gene that regulates relative prefrontal cortex dopamine availability, DBH, can identify those individuals who are less susceptible to bias in using computerized decision-aiding systems.

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.

Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.

Amino acid metabolomic analysis involved in flavor quality and cold tolerance in peach fruit treated with exogenous glycine betaine