Multistep Structural and Chemical Evaluation of Sugarcane Baggase, Pretreated With Alkali for Enhancing the Enzymatic Saccharification by Cellulase and Xylanase of the Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) Mix-Culture System

The enzymatic saccharification of sugarcane bagasse was significantly increased by alkali pretreatment under mild conditions. The effectiveness of different concentrations of alkali and acid pretreatment of sugarcane bagasse for improving the enzymatic saccharification of lignocellulose has been evaluated. The sugarcane bagasse was characterized to contain 39.52% celluloses, 25.63% hemicelluloses, and 30.36% lignin. After that, sugarcane bagasse was pretreated with 5 and 10% of H2SO4 and NaOH at 121°C for 60 min. FTIR, XRD, and SEM analyses also showed significant molecular and surface structure changes of the sugarcane bagasse with 10% NaOH. Maximum saccharification was 489.5 mg/g from 10% NaOH pretreatment followed by 322.75, 301.25, and 276.6 mg/g from 10% H2SO4, 5% NaOH, and 5% H2SO4, respectively, which were 55.1, 32.0, 27.1, and 20.6 times higher than the that of the control. Cellulase and xylanase produced by Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) were used to hydrolyze the pretreated bagasse, and the optimal condition was determined to be 30 h of the enzymatic reaction with the 3:1 ratio of enzymes under the temperature of 55°C, pH 5.0, and substrate concentration of 3%, leading to celluloses and hemicelluloses conversion in the enzymatic hydrolysis/saccharification that is more proficient.

Inhibition of the urea-urease reaction by the components of the zeolite imidazole frameworks-8 and the formation of urease-zinc-imidazole hybrid compound

In the past decade, much effort has been devoted to using chemical clock-type reactions in material design and driving the self-assembly of various building blocks. Urea-urease enzymatic reaction has chemical pH clock behavior in an unbuffered medium, in which the induction time and the final pH can be programmed by the concentrations of the reagents. The urea-urease reaction can offer a new alternative in material synthesis, where the pH and its course in time are crucial factors in the synthesis. However, before using it in any synthesis method, it is important to investigate the possible effects of the reagents on the enzymatic reaction. Here we investigate the effect of the reagents of the zeolite imidazole framework-8 (zinc ions and 2-methylimidazole) on the urea-urease reaction. We have chosen the zeolite imidazole framework-8 because its formation serves as a model reaction for the formation of other metal–organic frameworks. We found that, besides the inhibition effect of the zinc ions which is well-known in the literature, 2-methylimidazole inhibits the enzymatic reaction as well. In addition to the observed inhibition effect, we report the formation of a hybrid urease-zinc-2-methylimidazole hybrid material. To support the inhibition effect, we developed a kinetic model which reproduced qualitatively the experimentally observed kinetic curves.

Prospective Application of Aspergillus Species: Focus on Enzyme Production Strategies, Advances and Challenges

Fungal enzymes that catalyze different types of biochemical reactions play a significant role in modern industry by improving existing processes. Also, the use of enzymes to replace some traditional toxic chemical or mechanical approaches helps decrease energy demand and environmental pollution. However, enzymes must be able to compete commercially with relatively low-priced traditional approaches. Meeting economical and commercial feasibility criteria depends on a number of enzymatic properties including the specificity to the substrate, stability in industrial enzymatic reaction conditions and catalytic efficiency. Fungi used as an enzyme manufacture host should be appropriate for industrial scale fermentation. Aspergillus species are being developed as one of the best enzyme manufacture factories due to their capability to secrete high quantities of enzymes suitable for industrial applications. The industrial importance of Aspergillus species also includes the progress and commercialization of new products derived from genetically engineered modified strains. Hence, the main aim of this chapter investigation is to analyze the secreted and cellular proteins from Aspergillus species and their application in industries.

Polyamidoamine-Remdesivir Conjugate: Physical Stability and Cellular Uptake Enhancement

Nucleoside analogue antiviral remdesivir works by inhibiting the RNA-dependent RNA polymerase enzyme and terminating the viral replication. Currently, remdesivir is under a clinical trial for its activity against SARS-CoV-2. In the blood, remdesivir will undergo an enzymatic reaction to become monophosphate analogue form which is difficult to penetrate into the cell membrane. PAMAM (polyamidoamine) dendrimer is a good carrier to encapsulate remdesivir as a water-insoluble drug (0,339 mg/mL). Entrapment of remdesivir in the PAMAM cavity avoided remdesivir molecules to not undergo the enzymatic reactions. This study aimed to synthesize, characterize and evaluate cellular uptake of PAMAM-Remdesivir conjugate. PAMAM-Remdesivir was prepared with various stirring times (3, 6, 12, 24, and 48 hours). The conjugates were characterized to observe the size and particle distribution using Particle Size Analyzer, encapsulating efficiency using UV-Vis Spectroscopy, interaction between PAMAM and remdesivir particle using Fourier Transform Infrared Spectroscopy and cellular uptake of PAMAM-RDV using Fluorescence Microscope. The optimized stirring time of PAMAM-Remdesivir conjugate was 24 hours wich resulted the particles charge of + 23,07 mV of zeta potential, 1008 nm of particle size, 0,730 of PDI, and 69% entrapment efficiency. In addition, the FTIR analysis showed that remdesivir molecules successfully conjugated to PAMAM. Thus, through strring optimization time, the remdesivir molecules were successfully entrapped to PAMAM cavity. The cellular uptake in Vero Cell of PAMAM-RDV conjugated fluorescein isothiocyanate was observed with fluorescence microscope and had a stronger intensity than remdesivir only solution.

Fractal–Fractional Michaelis–Menten Enzymatic Reaction Model via Different Kernels

In this paper, three new models of fractal–fractional Michaelis–Menten enzymatic reaction (FFMMER) are studied. We present these models based on three different kernels, namely, power law, exponential decay, and Mittag-Leffler kernels. We construct three schema of successive approximations according to the theory of fractional calculus and with the help of Lagrange polynomials. The approximate solutions are compared with the resulting numerical solutions using the finite difference method (FDM). Because the approximate solutions in the classical case of the three models are very close to each other and almost matches, it is sufficient to compare one model, and the results were good. We investigate the effects of the fractal order and fractional order for all models. All calculations were performed using Mathematica software.

Epimerization of Deoxynivalenol by the Devosia Strain A6-243 Assisted by Pyrroloquinoline Quinone

Deoxynivalenol (DON) is a secondary metabolite produced by several Fusarium species that is hazardous to humans and animals after entering food chains. In this study, by adding cofactors, the Devosia strain A6-243 is identified as the DON-transforming bacteria from a bacterial consortium with the ability to biotransform DON of Pseudomonas sp. B6-24 and Devosia strain A6-243, and its effect on the biotransformation process of DON is studied. The Devosia strain A6-243 completely biotransformed 100 μg/mL of DON with the assistance of the exogenous addition of PQQ (pyrroloquinoline quinone) within 48 h and produced non-toxic 3-epi-DON (3-epi-deoxynivalenol), while Pseudomonas sp. B6-24 was not able to biotransform DON, but it had the ability to generate PQQ. Moreover, the Devosia strain A6-243 not only degraded DON, but also exhibited the ability to degrade 3-keto-DON (3-keto-deoxynivalenol) with the same product 3-epi-DON, indicating that DON epimerization by the Devosia strain A6-243 is a two-step enzymatic reaction. The most suitable conditions for the biodegradation process of the Devosia strain A6-243 were a temperature of 16–37 °C and pH 7.0–10, with 15–30 μM PQQ. In addition, the Devosia strain A6-243 was found to completely remove DON (6.7 μg/g) from DON-contaminated wheat. The results presented a reference for screening microorganisms with the ability of biotransform DON and laid a foundation for the development of enzymes for the detoxification of mycotoxins in grain and its products.

ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

Candida albicans phosphate transport, facilitating nucleotide sugar biosynthesis, contributes to cell wall stability.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

Growth and Phytase Activities of Bacillus subtilis IMV B-7023 During Cultivation with Sodium Phytate

Bacteria of the genus Bacillus are known for their ability to mineralize organic phosphorus compounds. Phytates constitute up to 60–80% of the total plant phosphorus and almost 50% of soil organic phosphorus. Phytates phosphorus is unavailable for plants. Bacillus can synthesize phosphatases both wide spectrum of action, and highly specific phytases that catalyze the hydrolysis of phytates. Therefore, the aim of this work was to study the growth and phytase activity of Bacillus subtilis IMV B-7023, which is the component of the ”Azogran” complex bacterial preparation for plant growing. Methods. The growth activity of bacteria was studied by cultivation methods, the phytase activity – by measuring the amount of phosphate released from sodium phytate during the enzymatic reaction. Results. It was shown that B. subtilis IMV B-7023 assimilated phytate as the source of phosphorus nutrition during cultivation in media with 0.5, 1.0 and 2.0 g/L of sodium phytate. The highest growth activity of these bacteria was observed after two days of cultivation in medium with 1.0 g/L of phytate. The number of bacteria was (3.91±0.32)×109 CFU/mL under these conditions. At the same time, B. subtilis IMV B-7023 demonstrated a low level of phytate assimilation as a source of carbon nutrition. Thus, after two days of cultivation the number of bacteria increased from (4.12±0.09)×106 CFU/mL to (1.07±0.07–3.11±0.51)×107 CFU/mL in the presence of 0.5–2.0 g/L phytate in the medium and the absence of another carbon source. It was determined that strain B. subtilis IMV B-7023 had phytase activity, the highest activity (221.85±0.12 U/g) was on the first day of their cultivation in medium with inorganic phosphates. It should be noted that B. subtilis IMV B-7023 phytase activity was lower during cultivating in medium with sodium phytate as a source of phosphorus nutrition, than in medium with inorganic phosphates. The obtained fact may be due to phytate hydrolysis by widespecific phosphatases. Higher rates of phytase activity obtained on the first and third days compared to the second and fourth days of bacterial cultivation may indicate the expression of phosphatases genes only in the period required for maximum bacterial development, in the absence of these proteins in the media. At the same time, the phytase activity of B. subtilis IMV B-7023 after 2 days cultivation in a media with 0.5 and 1.0 g/L of sodium phytate (194.80±0.15 U/g and 160.90±0.13 U/g, respectively) as the source of carbon and phosphorus was higher compared to the activity of bacteria on medium with inorganic phosphates (137.79±0.10 U/g). This may be caused by the synthesis of a larger number of highly specific phosphatases (phytases) in bacterial cells at the presence of only phytate in the medium as a substrate. Conclusions. B. subtilis IMV B-7023 strain is characterized by growth on nutrient medium with sodium phytate and phytase activity. Because they are soil microorganisms used as the component of the ”Azogran” complex bacterial preparation for plant growing, the ability to hydrolyze and assimilate phytate is important for functioning of this strain in the rhizosphere. The obtained results extend the understanding of B. subtilis IMV B-7023 influence on phosphorus nutrition and development of plants.

Pharmacological Investigation of CC-LAAO, an L-Amino Acid Oxidase from Cerastes cerastes Snake Venom

Snake venom proteins, which are responsible for deadly snakebite envenomation, induce severe injuries including neurotoxicity, myotoxicity, cardiotoxicity, hemorrhage, and the disruption of blood homeostasis. Yet, many snake-venom proteins have been developed as potential drugs for treating human diseases due to their pharmacological effects. In this study, we evaluated the use of, an L-amino acid oxidase isolated from Cerastes cerastes snake venom CC-LAAO, as a potential anti-glioblastoma drug, by investigating its in vivo and in vitro pharmacological effects. Our results showed that acute exposure to CC-LAAO at 1 and 2.5 µg/mL does not induce significant toxicity on vital organs, as indicated by the murine blood parameters including aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) activities, and creatinine levels. The histopathological examination demonstrated that only at high concentrations did CC-LAAO induce inflammation and necrosis in several organs of the test subjects. Interestingly, when tested on human glioblastoma U87 cells, CC-LAAO induced a dose-dependent apoptotic effect through the H2O2 generated during the enzymatic reaction. Taken altogether, our data indicated that low concentration of CC-LAAO may be safe and may have potential in the development of anti-glioblastoma agents.