Enzyme Activity Profiles Research Articles

Microbial enzymatic indices for predicting composting quality of recalcitrant lignocellulosic substrates

Three different enzymes alkaline phosphatase, Urease and Dehydrogenase were measured during this study to monitor the organic matter dynamics during semi-industrial composting of mixture A with 1/3 sludge+2/3 palm waste and mixture B with ½ sludge+1/2 palm waste. The phosphatase activity was higher for Mix-A (398.7 µg PNP g−1 h−1) than Mix-B (265.3 µg PNP g−1 h−1), while Mix-B (103.3 µg TPF g−1d−1) exhibited greater dehydrogenase content than Mix-A (72.3 µg TPF g−1 d−1). That could contribute to the dynamic change of microbial activity together with high amounts of carbonaceous substrates incorporated with the lignocellulosic. The gradual increase in the dehydrogenase from the compost Mix-A implies that high lignocellulosic substrate requires gradual buildup of dehydrogenase activity to turn the waste into mature compost. A higher pick of urease with a maximum activity of 151.5 and 122.4 µg NH4-N g−1 h−1 were reported, respectively for Mix-A and B. Temperature and pH could also influence the expression of enzyme activity during composting. The machine learning well predicted the compost quality based on NH3/NO3, C/N ratio, decomposition rate and, humification index (HI). The root mean square error (RMSE) values were 1.98, 1.95, 4.61%, and 4.1 for NH+3/NO−3, C/N ratio, decomposition rate, and HI, respectively. The coefficient of determination between observed and predicted values were 0.87, 0.93, 0.89, and 0.94, for the r NH3/NO3, C/N ratio, decomposition rate, and HI. Urease activity significantly predicted the C/N ratio and HI only. The profile of enzymatic activity is tightly linked to the physico-chemical properties, proportion of lignocellulosic-composted substrates. Enzymatic activity assessment provides a simple and rapid measurement of the biological activity adding understunding of organic matter transformation during sludge-lignocellulosic composting.

Epigenetic discovery by enzyme activity profiling.

Epigenetic discovery by enzyme activity profiling.

Expression study of Krabbe Disease GALC missense variants - Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels.

Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the GALC gene, which encodes for the enzyme galactosylceramidase (GALC). GALC is crucial for myelin metabolism. Functional deficiency of GALC leads to toxic accumulation of psychosine, dysfunction and death of oligodendrocytes, and eventual brain demyelination. To date, 46 clinically-relevant, pathogenic GALC missense mutations (MMs) have been identified in KD patients. These MMs are present in ∼70% of KD cases reported over 8 published studies between 1996 - 2019. However, the mechanisms by which these MMs lead to GALC functional deficiency and their correlations with clinical phenotype remain poorly understood. To address this, we generated a GALC -knockout human oligodendrocytic cell line (MO3.13/ GALC -KO) using CRISPR-Cas9 method to assess GALC function and GALC secretion. We evaluated 5 polymorphic and 31 clinically-relevant MM variants (MMVs) using transient expression assays. Our results showed that 26 MMVs, including 10 co-variants with p.I562T, reduced GALC activity by 92% - 100% compared to wild-type GALC (WT-GALC). MMVs from infantile-onset KD patients produced < 2% of WT activity, whereas those associated with juvenile- and adult-onset cases retained up to 7% of WT activity. Residual GALC activity was correlated with mature, lysosomal GALC protein levels (Pearson r = 0.93, P<0.0001). Many low-activity MMVs did not correspondingly impair GALC secretion. Twenty-one of the 26 low-activity MMVs showed a 21% - 100% reduction in sec-GALC levels, indicating varying degrees of GALC mis-trafficking among these variants. Importantly, GALC activity among MMVs strongly correlates with clinical disease severity, based on the age of symptom onset in patients with either homozygous MM (Pearson r = 0.98, P<0.0001, n = 7) or compound heterozygous (Pearson r = 0.94, P<0.0001, n = 12) MM-null mutation genotypes. Thus, our data suggests that GALC activity could serve as a prognostic disease indicator under specific experimental conditions. We further investigated the impact of pathogenic MMVs on psychosine accumulation, a key biomarker for KD. Psychosine levels were 21-fold higher in mock control cells compared to WT-GALC transfected cells (mock = 0.349 pmol/mg, WT-GALC = 0.016 pmol/mg), but negatively correlated with GALC activity among pathogenic MMVs (Pearson r = -0.63, P < 0.01, n = 15). Although psychosine levels were higher in most MMVs associated with infantile-onset KD, no significant correlations with clinical onset were detected. Overall, our study provides a comprehensive quantitative analysis of the functional deficits and mis-trafficking associated with clinically-relevant GALC MMVs, enhancing our understanding of the molecular genetics and genotype-phenotype correlations of the GALC gene in Krabbe disease.

Safety Assessment and Evaluation of Probiotic Potential of Lactobacillus bulgaricus IDCC 3601 for Human Use

Lactic acid bacteria (LAB) are probiotic microorganisms widely used for their health benefits in the food industry. However, recent concerns regarding their safety have highlighted the need for comprehensive safety assessments. In this study, we aimed to evaluate the safety of L. bulgaricus IDCC 3601, isolated from homemade plain yogurt, via genomic, phenotypic, and toxicity-based analyses. L. bulgaricus IDCC 3601 possessed a single circular chromosome of 1,865,001 bp, with a GC content of 49.72%, and 1910 predicted coding sequences. No virulence or antibiotic resistance genes were detected. Although L. bulgaricus IDCC 3601 exhibited antibiotic resistance to gentamicin and kanamycin, this resistance is an intrinsic feature of this species. L. bulgaricus IDCC 3601 did not produce biogenic amines and did not exhibit hemolytic activity. Phenotypic analysis of enzyme activity and carbohydrate fermentation profiles revealed the metabolic features of L. bulgaricus IDCC 3601. Moreover, no deaths or abnormalities were observed in single-dose oral toxicity tests, suggesting that L. bulgaricus IDCC 3601 has no adverse effect on human health. Finally, L. bulgaricus IDCC 3601 inhibited the growth of potential carbapenem-resistant Enterobacteriaceae. Therefore, our results suggest that L. bulgaricus IDCC 3601 is a safe probiotic strain for human consumption.

An Enhanced Interaction of Graft and Exogenous SA on Photosynthesis, Phytohormone, and Transcriptome Analysis in Tomato under Salinity Stress.

Salt stress can adversely affect global agricultural productivity, necessitating innovative strategies to mitigate its adverse effects on plant growth and yield. This study investigated the effects of exogenous salicylic acid (SA), grafting (G), and their combined application (GSA) on various parameters in tomato plants subjected to salt stress. The analysis focused on growth characteristics, photosynthesis, osmotic stress substances, antioxidant enzyme activity, plant hormones, ion content, and transcriptome profiles. Salt stress severely inhibits the growth of tomato seedlings. However, SA, G, and GSA improved the plant height by 22.5%, 26.5%, and 40.2%; the stem diameter by 11.0%, 26.0%, and 23.7%; the shoot fresh weight by 76.3%, 113.2%, and 247.4%; the root fresh weight by 150.9%, 238.6%, and 286.0%; the shoot dry weight by 53.5%, 65.1%, and 162.8%; the root dry weight by 150.0%, 150.0%, and 166.7%, and photosynthesis by 4.0%, 16.3%, and 32.7%, with GSA presenting the most pronounced positive effect. Regarding the osmotic stress substances, the proline content increased significantly by more than 259.2% in all treatments, with the highest levels in GSA. Under salt stress, the tomato seedlings accumulated high Na+ levels; the SA, G, and GSA treatments enhanced the K+ and Ca2+ absorption while reducing the Na+ and Al3+ levels, thereby alleviating the ion toxicity. The transcriptome analysis indicated that SA, G, and GSA influenced tomato growth under salt stress by regulating specific signaling pathways, including the phytohormone and MAPK pathways, which were characterized by increased endogenous SA and decreased ABA content. The combined application of grafting and exogenous SA could be a promising strategy for enhancing plant tolerance to salt stress, offering potential solutions for sustainable agriculture in saline environments.

Benzophenone-4 inhibition in marine diatoms: Physiological and molecular perspectives

Benzophenone-4 (BP-4), a widely utilized organic ultraviolet (UV) filter, is recognized as a pseudo-persistent contaminant in aquatic environments. To elucidate the effects and mechanisms of BP-4 on marine diatoms, an investigation was conducted on the growth rate, photosynthetic pigment content, photosynthetic parameters, antioxidant enzyme activity, malondialdehyde (MDA) levels, cellular structure, and transcriptome profile of the model species, Phaeodactylum tricornutum. The results showed a pronounced inhibition of algal growth upon exposure to BP-4, with a 144 h-EC50 value of 201 mg·L−1. In addition, BP-4 exposure resulted in a significant reduction in biomass, disruption of cell membrane integrity, and increased MDA accumulation, with levels escalating 3.57-fold at 125 mg·L−1 of BP-4. In the BP-4-treated samples, 1556 differentially expressed genes (DEGs) were identified, of which 985 were upregulated and 571 were downregulated. Gene ontology and KEGG pathway enrichment analysis revealed that the carbon fixation and carbon metabolism processes in P. tricornatum were disrupted in response to BP-4 exposure, along with excessive reactive oxygen species (ROS) production. The upregulation of genes associated with photosynthetic pigment (chlorophyll and carotenoids) synthesis, phospholipid synthesis, ribosome biogenesis, and translation-related pathways may be regarded as a component of P. tricornatum's tolerance mechanism towards BP-4. These results provide preliminary insights into the toxicity and tolerance mechanisms of BP-4 on P. tricornatum. They will contribute to a better understanding of the ecotoxicological impacts of BP-4 on the marine ecosystem and provide valuable information for elimination of BP-4 in aquatic environment by bioremediation.

Effect of chokeberry pomace as a feed additive for high-producing dairy goats on oxidative stress parameters and quality of milk

Abstract Introduction Chokeberry pomace, rich in polyphenolic compounds, holds potential to be a valuable feed additive for enhancing the antioxidative capacity and overall quality of milk. This study explores the impact of dietary inclusion of chokeberry pomace on oxidative stress parameters and other milk quality parameters in high-producing dairy goats. Material and Methods Twenty-seven goats were allocated into three groups: a control group provided standard feed and two experimental groups provided feed supplemented with 15 g or 30 g of chokeberry pomace per kilogram. Milk samples were analysed for physicochemical traits, a range of enzyme activities and antioxidant properties. Results Supplementation with chokeberry pomace significantly reduced milk fat content, enhanced antioxidative properties and increased most of the quantified enzyme activities. Total polyphenol content and reduced glutathione levels were significantly higher in the supplemented groups, correlating with improved antioxidative potential of the milk. Conclusion Chokeberry pomace in goat diets enhances milk’s antioxidative properties and upregulates its enzymatic activity profile, suggesting a potential strategy to improve the nutritional quality and health benefits of goat milk. The study underscores the utility of chokeberry pomace as a feed additive that might not only benefit animal health but also contribute to enhanced milk quality.

Arbuscular mycorrhizal fungi alter carbon metabolism and invertase genes expressions of Populus simonii × P. nigra under drought stress.

Arbuscular mycorrhizal fungi (AMF) play a crucial role in regulating the allocation of carbon between source and sink tissues in plants and in regulating their stress responses by changing the sucrose biosynthesis, transportation, and catabolism in plants. Invertase, a key enzyme for plant development, participates in the response of plants to drought stress by regulating sucrose metabolism. However, the detailed mechanisms by which INV genes respond to drought stress in mycorrhizal plants remain unclear. This study examined the sugar content, enzyme activity, and expression profiles of INV genes of Populus simonii × P. nigra (PsnINVs) under two inoculation treatments (inoculation or non-inoculation) and two water conditions (well-watered or drought stress). Results showed that under drought stress, AMF up-regulated the expressions of PsnA/NINV1, PsnA/NINV2, PsnA/NINV3, and PsnA/NINV5 in leaves, which may be related to the enhancement of photosynthetic capacity. Additionally, AMF up-regulated the expressions of PsnA/NINV6, PsnA/NINV10, and PsnA/NINV12 in leaves, which may be related to enhancing osmotic regulation ability and drought tolerance.

Variations in quality attributes of pulsed light-treated table grape juice during refrigerated storage (4°C) and ambient conditions (25°C).

Pulsed light (PL) pasteurization is being explored as a substitute for the conventional thermal pasteurization of juices in recent times due to better retention of nutrients and overall quality. However, the long-term stability of the PL-pasteurized juice must be investigated to promote its application by the industry. The effect of PL treatment (effective fluence of 1.15 J·cm-2) and thermal treatment (90°C for 60 s) on microbial quality, enzyme activity, bioactive compounds, sensory acceptance, and color profile of table grape juice during storage at 4 and 25°C was investigated in this study. The PL pasteurization enhanced the microbial shelf-life of the juice (<6 log10cfu·mL-1) from 5 to 35 days at 4°C. The PL and thermally-pasteurized juice demonstrated a shelf-life of only 10 days when stored at 25°C. The total soluble solids and titratable acidity did not alter significantly throughout the storage period. The peroxidase, polyphenol oxidase, and pectin methylesterase activities were below 10% for the PL and thermally-treated beverage when stored at 4°C. The sensory acceptability of the PL-pasteurized juice after 35 days of refrigerated storage (6.9±0.3) was close to the untreated juice (7.2±0.3) and greater than thermally-treated juice (6.2±0.2). After the 35th day of storage at 4°C, PL-treated grape juice retained 55%, 12%, and 15.3% more phenolics, flavonoids, and antioxidant capacity, respectively, than the thermally-pasteurized juice. Hence, PL pasteurization can effectively prolong the shelf-life of table grape juice while achieving microbial and enzymatic stability, along with high sensory and nutritional appeal. PRACTICAL APPLICATION: Exploring non-thermal methods like pulsed light (PL) pasteurization as a substitute for conventional thermal methods is gaining recognition for its ability to retain nutrients and improve overall juice quality. However, the industry's adoption depends on understanding the shelf-stability of PL-pasteurized juice. This study specifically investigates the practical applications of PL treatment in comparison with conventional thermal treatment in enhancing microbial safety and enzymatic stability in table grape juice. The findings contribute insights into optimizing the shelf life of table grape juice and preserving its quality, supported by microbial, enzymatic, and sensory evaluations.

An evanescent waves sensor based on polydopamine-gold coated chalcogenide tapered fiber for enzyme activity profiling

In this research, we demonstrate a high-sensitivity tapered fiber (TF) sensor made of As2S3 chalcogenide glass coated with polydopamine (PDA) and gold nanoparticles (AuNPs). The presence of in-situ grown AuNPs on the PDA-coated TF surface via the reduction of Au ions by catechol groups in PDA, facilitated efficient light coupling with evanescent waves, thereby improving the IR absorption of molecules. The sensor was utilized to measure the activity of the glucose oxidase enzyme as a point-of-concept analyte. This process of enzyme catalyzing the conversion of glucose to hydrogen peroxide and Gluconic acid can be recorded by the sensor. In investigating the enzyme activity and D-glucose oxidation kinetics, we monitored the intensity of the Gluconic acid band at 5.764 μm, which arises from the C=O stretching vibration, as a function of time. The estimated enzyme activity using the PDA/AuNPs-coated TF sensor was 103.66 ± 2.87 Umg−1 when the enzyme activity of the sample was 100 Umg−1 which is consistent with standard value. The Lineweaver–Burk plot yielded the following kinetic constants: Km = 16.10 ± 1.63 mM and Vmax = 93.72 ± 12.71 μmol/min. Results suggest that this sensor has great application potential in various enzyme activity applications.

Innovative Colorimetric NQO1 Detection Strategy via Substrate Competitive and Biomimetic Cascade Reactions with a Highly Active NADH Oxidase Mimic.

NAD(P)H: quinone oxidoreductase-1 (NQO1) plays critical roles in antioxidation and abnormally overexpresses in tumors. Developing a fast and sensitive method of monitoring NQO1 will greatly promote cancer diagnosis in clinical practice. This study introduces a transformative colorimetric detection strategy for NQO1, harnessing an innovative competitive substrate mechanism between NQO1 and a new NADH oxidase (NOX) mimic, cobalt-nitrogen-doped carbon nanozyme (CoNC). This method ingeniously exploits the differential consumption of NADH in the presence of NQO1 to modulate the generation of H2O2 from CoNC catalysis, which is then quantified through a secondary, peroxidase-mimetic cascade reaction involving Prussian blue (PB) nanoparticles. This dual-stage reaction framework not only enhances the sensitivity of NQO1 detection, achieving a limit of detection as low as 0.67 μg mL-1, but also enables the differentiation between cancerous and noncancerous cells by their enzymatic activity profiles. Moreover, CoNC exhibits exceptional catalytic efficiency, with a specific activity reaching 5.2 U mg-1, significantly outperforming existing NOX mimics. Beyond mere detection, CoNC serves a dual role, acting as both a robust mimic of cytochrome c reductase (Cyt c) and a cornerstone for enzymatic regeneration, thereby broadening the scope of its biological applications. This study not only marks a significant step forward in the bioanalytical application of nanozymes but also sets the stage for their expanded use in clinical diagnostics and therapeutic monitoring.

Impact of silymarin-supplemented cookies on liver enzyme and inflammatory markers in non-alcoholic fatty liver disease patients.

Nonalcoholic fatty liver disease (NAFLD) is a growing public health concern characterized by fat accumulation and severe disorders like nonalcoholic steatohepatitis (NASH), which are influenced by obesity, inflammatory processes, and metabolic pathways. This research investigates the potential of silymarin-supplemented cookies in managing NAFLD by evaluating their impact on liver enzyme activity, inflammatory markers, and lipid profiles. A clinical trial in Lahore, Pakistan, involved 64 NAFLD patients. Participants were divided into placebo and three treatment groups, with the latter receiving silymarin-supplemented cookies for 3 months. The study assessed liver enzyme levels and inflammatory markers, at baseline and after the intervention, utilizing statistical analyses to evaluate differences. The lipid profile and renal function test (RFT) were also measured at baseline and after 3 months in each group for safety assessment. After 3 months, the treatment groups indicated more significant decreases in liver enzymes compared to the placebo group (p ≤ .05). Treatment 3 showed significant reductions in alanine aminotransferase (ALT) (64.39-49.38 U/L) and aspartate aminotransferase (AST) (61.53-45.38 U/L). Treatment 3 also showed improvements in alkaline phosphatase (ALP) levels and the AST/ALT ratio. Additionally, the treatment group demonstrated a significant reduction in inflammatory markers. Treatment 3 showed a significant decrease in C-reactive protein (CRP) (6.32-3.39 mg/L) and erythrocyte sedimentation rate (ESR) (38.72-23.86 mm/h), indicating that individuals with NAFLD may benefit from the intervention's potential benefits in lowering inflammation. The study revealed that an intervention significantly improved the inflammatory markers, liver enzymes, and lipid profiles of NAFLD participants, suggesting potential benefits for liver health.

FEATURES OF THE COURSE OF NON-ALCOHOLIC FATTY LIVER DISEASE INTYPE 2 DIABETES MELLITUS AND IMPAIRED GLUCOSE TOLERANCE

Background. Non-alcoholic fatty liver disease and type 2 diabetes mellitus or impaired glucose tolerance are common diseases with a high risk of developing cardiovascular diseases, the leading cause of disability and death. Our aim is to develop new methods for screening, prevention, and treatment of cardiovascular diseases in patients with non-alcoholic fatty liver disease, type 2 diabetes mellitus, and impaired glucose tolerance. Methods and methods. This study is single-center, open-label, uncontrolled, diagnostic study. Between 2023 and February 2024, 216 patients with cardiovascular diseases and concomitant non-alcoholic fatty liver disease, type 2 diabetes, and impaired glucose tolerance were selected at Heart Center “University Medical Center” Corporate Fund. Results. All examined patients with cardiovascular diseases and concomitant non-alcoholic fatty liver disease showed increased liver enzyme activity. Blood lipid profile indicators were significantly higher than optimal levels for patients with cardiovascular diseases. There was also a significant increase in liver enzymes, C-reactive protein, triglycerides, and lipoproteins in patients with cardiovascular diseases and non-alcoholic fatty liver disease combined with type 2 diabetes and impaired glucose tolerance. Conclusion. Liver enzyme activity, C-reactive protein, glucose, and lipid profile analysis showed significant increases in these indicators in patients with cardiovascular diseases and non-alcoholic fatty liver disease, especially in the presence of type 2 diabetes and impaired glucose tolerance. All examined patients showed increased liver enzyme activity and lipid levels, indicating the impact of these diseases on the liver and metabolism

Connecting genes to whole plants in dilution effect of target-site ALS inhibitor resistance of Schoenoplectiella juncoides (Roxb.) Lye (Cyperaceae)

This study focuses on dilution effect of target-site resistance (TSR) to acetolactate synthase (ALS) inhibitors in Schoenoplectiella juncoides, which harbors two ALS genes, ALS1 and ALS2. We assessed gene expression, enzyme activity, and whole-plant resistance profiles across four S. juncoides lines: the susceptible line, the parental resistant lines with a homozygous mutation in either ALS1 or ALS2, and the bred progeny line with homozygous mutations in both ALS1 and ALS2. Gene expression and enzyme function showed a proportional relationship that the expression ratios of ALS1 to ALS2, approximately 70:30, were consistent with the functional ratio predicted by the double-sigmoidal plateau positions observed in enzyme assays. However, at the whole-plant level, resistance did not correlate to the putative abundance of susceptible enzyme, but the parental lines showed similar resistance to each other despite different enzyme-level resistances. This suggests a non-proportional mechanism in the reflection of physiological enzymatic profiles to whole-plant resistance profiles. These findings highlight the complexity of herbicide resistance and the need for further research to understand the mechanisms that influence resistance outcomes. Understanding these relationships is essential for developing strategies to manage herbicide resistance effectively.

Sustainable combination of ionic liquid and deep eutectic solvent for protecting and preserving of the protein structure: The synergistic interaction of enzymes and eco-friendly hybrid ionic fluids

Hybrid ionic fluids (HIFs) are one of the emerging and fascinating sustainable solvent media, a novel environment-friendly solvent for biomolecules. The HIFs have been synthesized by combining a deep eutectic solvent (DES), an ionic liquid (IL) having a common ion. The stability and activity of hen's egg white lysozyme (Lyz) in the presence of a recently designed new class of biocompatible solvents, HIFs have been explored by UV–visible, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) along with dynamic light scattering (DLS) measurements. This work emphasizes the effect of DES synthesized by using 1:2 choline chloride and glycerol [Glyn], ILs (1-butly-3-methylimidazolium chloride [BMIM]Cl and choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moving forward, we also studied the secondary structure, thermal stability and enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5) M of [BMIM]Cl, [Chn][Ac] ILs, [Glyn] DES and [Glyn][BMIM]Cl (hybrid ionic fluid1) as well as [Glyn][Chn][Ac] (hybrid ionic fluid2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz, whereas the stability and activity are increased by DES and are maintained by HIFs at all the studied concentrations. Overall, the experimental results studied elucidate expressly that the properties of Lyz are maintained in the presence of hybrid ionic fluid1 while these properties are intensified in hybrid ionic fluid2. This work has elucidated expressly biocompatible green solvents in protein stability and functionality due to the alluring properties of DES, which can counteract the negative effect of ILs in HIFs.

Biochemical Profiling and Characterization of Mesorhizobium ciceri Isolates from Chickpea Root Nodules: A Comprehensive Review

Mesorhizobium ciceri is a pivotal bacterium in the nitrogen fixation process of chickpea (Cicer arietinum), playing a crucial role in enhancing soil fertility and crop productivity. This comprehensive review delves into the biochemical profiling and characterization of M. ciceri isolates from chickpea root nodules. It details the methodologies employed for isolating and identifying these bacterial isolates, encompassing morphological, biochemical, and molecular techniques. The review highlights various biochemical assays, including carbon source utilization, enzyme activity, and antibiotic resistance profiles, alongside genetic and phenotypic characterizations through 16S rRNA sequencing, multilocus sequence typing, and genomic fingerprinting and explores the implications of these findings for sustainable agriculture, focusing on biofertilizer development, soil health, and improved crop management. By providing an in-depth understanding of M. ciceri‘s role in chickpea symbiosis, this review aims to enhance strategies for sustainable agricultural practices and legume crop productivity.

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity.

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver of the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being >1000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, and some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops and changes to their conformations, as revealed by a suite of crystal structures. This suggests that a specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining the enzyme activity profile.

Evaluating efficacy of Pseudomonas sp. EN-4 to lower the toxic potential of 4-bromophenol and assessing its competency in simulated microcosm

An indigenous bacterium Pseudomonas sp. EN-4 had been reported earlier for its ability to co-metabolise 4-bromophenol (4-BP), in presence of phenol (100 mg/L) as co-substrate. The present study was undertaken to validate the efficacy of biotransformation by comparing the toxicity profiles of untreated and EN-4 transformed samples of 4-BP, using both plant and animal model. The toxicity studies in Allium cepa (A. cepa) indicated to lowering of mitotic index (MI) from 12.77% (water) to 3.33% in A. cepa bulbs exposed to 4-BP + phenol, which reflects the cytotoxic nature of these compounds. However, the MI value significantly improves to 11.36% in its biologically treated counterpart, indicating normal cell growth. This was further supported by significant reduction in chromosomal aberrations in A. cepa root cells exposed to biologically treated samples of 4-BP as compared to untreated controls. The oxidative stress assessed by comparing the activity profiles of different marker enzymes showed that the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) were reduced by 56%, 72%, and 37% respectively, in EN-4 transformed samples of 4-BP + phenol compared to its untreated counterpart. Similar trends were evident in the comet assay of fish (Channa punctatus) blood cells exposed to untreated and biologically treated samples of 4-BP. The comparative studies showed significant reduction in tail length (72.70%) and % tail intensity (56.15%) in fish blood cells exposed to EN-4 treated 4-BP + phenol, compared to its untreated counterpart. The soil microcosm studies validated the competency of the EN-4 cells to establish and transform 4-BP in soil polluted with 4-BP (20 mg/kg) and 4-BP + phenol (20 + 100 mg/kg). The isolate EN-4 achieved 98.08% transformation of 4-BP in non-sterile microcosm supplemented with phenol, indicating to potential of EN-4 cells to establish along with indigenous microflora.

Metabolic Rate Suppression and Maintenance of Flight Muscle Metabolic Capacity during Diapause in Bumble Bee (Bombus impatiens) Queens.

AbstractThe common eastern bumble bee (Bombus impatiens) queens endure cold winter months by entering a diapause state. During this overwintering period, these animals use stored energy reserves while maintaining a low metabolic rate. This study investigates changes in the metabolic rate of bumble bee queens during diapause-like laboratory conditions and the potential reorganization of the flight muscle metabolic properties during this period. We first confirmed the hypometabolic state of queens during diapause in the laboratory, which lowered their resting metabolic rate to less than 5% of normal resting values. Body mass decreased during diapause, body composition changed where carbohydrates decreased initially, and later protein declined, with a similar trend for lipid content. Using cellular respirometry, we determined the capacity of the flight muscle cells of bumble bee queens to use various metabolic fuels and whether this capacity changes during the progression of diapause to favor stored lipid-derived substrates. Queens showed a low capacity to oxidize the amino acid proline, compared with workers, and their capacity to oxidize all metabolic substrates did not change during a 4-mo diapause period in the laboratory. We also show no detectable ability to oxidize fatty acid by flight muscle mitochondria in this species. The metabolic properties of flight muscle tissue were further characterized using metabolic enzyme activity profiles showing little change during diapause, indicating that profound metabolic suppression is induced without major changes in muscle metabolic phenotypes. Overall, B. impatiens queens undergo diapause while maintaining flight muscle capacity under the conditions used.

Evaluating mechanism of banana pseudo-stem retting using seawater: A cost-effective surface pre-treatment approach

Retting has been employed to extract natural fibers from agricultural wastes as a biological and cost-effective approach for centuries. With its global abundance, banana pseudo-stem is a promising agro-waste for lignocellulosic fiber extraction. In this study, fibers were extracted from the pseudo-stems after being pre-treated under four conditions using seawater at room temperature for up to 35 d Bacterial isolation from the fresh seawater sample and screening for ligninolytic ability were conducted. Bacterial load as well as laccase and manganese peroxidase enzyme activity profile assay during the retting duration were analyzed. Fourier transform infrared (FT-IR) and X-day diffraction (XRD) analyses were also examined for both pre-treated and untreated extracted fibers. The results shows that six out of the eight bacterial isolates had the ability to degrade lignin. The treatments (Raw stem + Raw seawater) and (Autoclaved stem + Raw seawater) recorded the highest viable bacterial load of 9.24 × 102 and 4.46 × 102 CFU, respectively, on the 14th day of the retting process. Additionally, the highest laccase and manganese peroxidase enzymes activity was recorded for (Raw stem + Raw seawater) and (Autoclaved stem + Raw seawater) treatments in the second to the third week. The FT-IR spectra of the pre-treated fibers revealed relative reductions in peaks attributed to polysaccharides and other amorphous substances for all retting conditions. The XRD diffractogram revealed that the crystallinity index (CI) of pre-treated fibers increased in all seawater retting treatment conditions. However, the CI for fibers pre-treated under enzymatic conditions were enhanced even after five weeks. Sequence analysis for selected bacterial isolates showed homology to sequences of Bacillus velezensis, Shewanella sp. L8–5, and Citrobacter amalonaticus and Bacillus subtilis j8 strain. From these findings, it was suggested that physical, biological, and chemical actions were collectively involved in the seawater retting process of banana pseudo-stems.