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Background: New potential biological targets prediction through inverse molecular docking technique is an another smart strategy to forecast the possibility of compounds being biologically active against various target receptors. Objectives: In this case of designed study, we screened our recently obtained novel acetylinic steroidal biotransformed products [(1) 8-β-methyl-14-α-hydroxy∆4tibolone (2) 9-α-Hydroxy∆4 tibolone (3) 8-β-methyl-11-β-hydroxy∆4tibolone (4) 6-β-hydroxy∆4tibolone, (5) 6-β-9-α-dihydroxy∆4tibolone (6) 7-β-hydroxy∆4tibolone) ] from fungi Cunninghemella Blakesleana to predict their possible biological targets and profiling of ADME properties. Method: The prediction of pharmacokinetics properties membrane permeability as well as bioavailability radar properties were carried out by using Swiss target prediction, and Swiss ADME tools, respectively these metabolites were also subjected to predict the possible mechanism of action along with associated biological network pathways by using Reactome data-base. Results: All the six screened compounds possess excellent drug ability criteria, and exhibited exceptionally excellent non inhibitory potential against all five isozymes of CYP450 enzyme complex, including (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) respectively. All the screened compounds are lying within the acceptable pink zone of bioavailability radar and showing excellent descriptive properties. Compounds [1-4 & 6] are showing high BBB (Blood Brain Barrier) permeation, while compound 5 is exhibiting high HIA (Human Intestinal Absorption) property of (Egan Egg). Conclusion: In conclusion, the results of this study smartly reveals that in-silico based studies are considered to provide robustness towards a rational drug designing and development approach, therefore in this way it helps to avoid the possibility of failure of drug candidates in the later experimental stages of drug development phases.

This study aimed to explore the optimum conditions for the ultrasonic-enzyme-assisted extraction of comfrey (Symphytum officinale L.) polysaccharides (SOP) by the response surface methodology (RSM). The optimum extraction conditions included an extraction time of 62 min, an enzyme complex dose of 1.1 %, a liquid-to-solid ratio of 21 mL/g, and a pH value of 6. A SOP yield of 20.32 % was achieved. The digestion and fermentation behaviors of SOP in vitro were determined. A digestion test was performed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). A fermentation model was prepared with the cecal contents of chickens. The results showed that SGF (2.15 ± 0.09 %) and SIF (1.08 ± 0.06 %) were ineffective in the digestion of SOP. During fermentation, the pH values of the culture medium were lower than the initial pH (7.75 ± 0.03). SOP (47.06 ± 1.79 %) was used by the chicken cecal microbiota, which produced 34.12 ± 0.79 mg/mL short-chain fatty acids at 48 h. Moreover, the composition of the chicken cecal microbiota could be regulated by SOP during fermentation. The relative abundance of Lactobacillus at the genus level was improved. Therefore, SOP might have the potential to serve as a prebiotic in chicken production.

Microorganisms including Bacillus can produce signal molecules such as surfactin, resulting in the variation of membrane potential to trigger quorum sensing such as biofilm formation and sporulation in response to the environment stresses. However, biosynthesis of surfactin requires multiple resources such as huge enzyme complex, amino acids, fatty acids, and energy. Insufficient resources in the natural soil environment restrain biosynthesis of surfactin. When surfactin is inadequate, cations in soil might serve as substitutes to regulate quorum sensing. Our results showed that both surfactin and cations could lead to the variation of membrane potential, thus providing signals to trigger the quorum sensing such as growth, biofilm formation, and sporulation in Bacillus amyloliquefaciens. Neither KinC nor Abh was essential for surfactin or cations to trigger quorum sensing. The cation signaling pathway is only partially dependent on Spo0A, but the surfactin signaling pathway is fully dependent on this global regulator. Compared to surfactin, cations are less effective in promoting biofilm formation, but more effective to trigger sporulation in B. amyloliquefaciens. This study reveals a pathway through which cations regulate the quorum sensing in B. amyloliquefaciens in the case of insufficient surfactin in environment.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

Marine macroalgae are potential renewable feedstocks for valuable biomaterials. Among them, alginate is a primary component in brown algae that can be nonenzymatically converted and enzymatically degraded by alginate lyases to 4-deoxy- l -erythro-5-hexoseulose uronic acid (DEH). Here, we constructed alginolytic enzyme complexes comprising two different alginate lyases for synergistic alginate degradation. The complexes showed good thermostability with 60% of the residual activity at high temperature (60 °C). Furthermore, they produced 0.85 and 0.18 mg/mL DEH from alginate and natural brown algae as substrates, respectively. The enzyme complex successfully decomposed brown algal biomass, resulting in a 3.15-fold improvement in DEH when compared to free enzymes. The Ralstonia eutropha strain with alginolytic enzyme complexes on the cell surface showed higher Polyhydroxybutyrate (PHB) production and produced 2.58 g/L PHB from alginate. After the use of alginate, remaining biomass such as fucoidan and laminaran can also be used in the future for high value ingredients in nutritional, medical device, skincare and dermatological products. These results demonstrate that it is possible to create more efficient strategies for producing biodegradable PHB and functional polysaccharides from brown algal substrates.

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Drugs show specific pharmacodynamic properties and a plethora of adverse drug reactions depending on the genetic makeup of their users. Pharmacogenomics explains the variations of effects of drugs in humans depending on their race, ethnicity, age and gender. It has been extensively reported in the literature that variations in drug metabolism is associated with genetic polymorphism. Non-steroidal anti-inflammatory drugs (NSAIDs) are prescribed for pain management in arthritis. Cytochrome P450 (CYP) 2C9 is responsible for the metabolism of a wide range of drugs including the NSAIDs. The gene encoding for this enzyme is found in different polymorphic forms. People with CYP variants, CYP2C9*2 and CYP2C9*3 have been found to exhibit marked differences in NSAID metabolism and clearance compared to individuals with the wild type CYP gene (CYP2C9*1). There are several reports indicating epigenetic regulation of the CYP promoter that influences expression and functionality of this enzyme complex. Complications like adverse drug reactions involving gastro-intestinal bleeding are often fatal in NSAID treated patients with genetic polymorphism. In this review, complications associated with the use of NSAIDs and CYP polymorphism including drugs that are under investigation to bypass CYP polymorphism-linked adverse drug reactions, are discussed.

Mammalian innate immune response to virus infection is meditated by many cell-intrinsic pathways. RNA viruses, such as Sendai virus, which replicate in the cytoplasm, trigger the RIG-I-like receptor pathway, which activates the transcription factor, IRF3. Activated IRF3 translocates to the nucleus and induces transcription of the genes which encode interferons, the major antiviral cytokines. Interestingly, IRF3 activates another interferon-independent antiviral pathway, called RIG-I induced pathway of apoptosis (RIPA). For activating RIPA, IRF3 translocates from the cytoplasm to mitochondria. RIPA requires linear polyubiquitination of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 binds to Bax and translocates it to mitochondria causing the release of Cytochrome C, activation of caspases and apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, inhibits RIPA by deubiquitinating IRF3. Ablation of Otulin expression enhanced RIPA and its overexpression inhibited RIPA. In virus-infected cells, to overcome Otulin-mediated inhibition, RIPA actively degrades Otulin. This degradation required sequential actions of RIPA-activated Caspase 3 and proteasomes. Caspase 3 cleaved Otulin at D31; the D31A mutant was not cleaved at all. The caspase-cleaved fragment was totally degraded by proteasomes, which was preceded by its K48-linked ubiquitination. Mass spectrometric analysis of Otulin identified K64 and K197 as the ubiquitinated residues. Otulin interacted with LUBAC after virus infection and the E3-ubiquitin ligase, HOIP, a component of LUBAC, ubiquitinated Otulin to trigger its proteasome-mediated degradation. To assess the impact of Otulin degradation on RIPA-mediated antiviral action, we expressed, in Otulin-ablated cells, a non-degradable mutant of Otulin, in which D31, K64 and K197 had been mutated. The cells expressing the Otulin mutant were less susceptible to virus-induced apoptosis, because RIPA was less active, and consequently virus replication was more robust. Thus, our study has revealed an important positive feedback loop of RIPA.

The crystal structure of l -lactate oxidase in complex with l -lactate was solved at a 1.33 A resolution. The electron density of the bound l -lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l -lactate was bound to the FMN and an additional active site within the enzyme complex. l -lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l -lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

Specific features of the enzymatic degradation of chitosan with depolymerization degrees (DD) of 85 and 50% by microbial chitinases and chitosanases were studied in terms of the conversion degree, molecular-weight distribution (MWD), and antimicrobial activity of the generated reaction products. The enzyme complex produced by the strain B. thuringiensis B-387, which is characterized by high chitosanase production (>4.5 U mL–1), degraded the polymer to soluble, low molecular weight chitooligosaccharides (CHOs, Mw ≤ 2 kDa), along with a minor yield (~5%) of alkali-precipitated oligochitosans (Mw 2–16 kDa). The hydrolytic complexes produced by B. atrophaeus IB-33-1 and Cohnella sp. IB-P192, which mainly comprise chitinases (0.3–0.5 U mL–1), demonstrated the lowest rate and degree of chitosan (DD 85%) hydrolysis. The selection of an enzyme : substrate ratio in the range of 2–5 units/g (based on chitosanase) made it possible to reduce the hydrolysis depth of the initial polymer and to increase the yield of oligochitosans with a Mw of ~15–17 kDa to 30% for chitosan (DD 85%) depolymerization by the enzyme complex from B. thuringiensis B-387. The decrease in the bactericidal and fungicidal effect of the oligomers that formed during the destruction of chitosan with varying deacetylation degrees (DD 85% and 50%) by enzyme complexes displaying high chitosanase activity was, as a rule, more distinct than that achieved with the use of chitinases. However, in some cases, there were both nonspecific and specific enhancements of the antimicrobial action of hydrolytic products in comparison with the initial polymer, which was determined by individual sensitivity of bacterial and micromycetes strains.

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications.

empeh is an Indonesian traditional fermented food with rich nutrition and bioactive components. Rhizopus sp,especially Rhizopus oligosporus), lactic acid bacteria (Lactobacillus sp.), and yeast aremicroorganisms involved in Tempeh fermentation. An interesting offer of Bioinformatics (in silico method) as a supporting tool in molecular biology studies has emerged, such as in protein cleavage. This study utilized PeptideCutter application on ExPASyBioinformatics portal (https://web.expasy.org/peptide_cutter/) to cleave soy proteins glycinin G1, G2, G3, G4,G5,-conglycinin-chain,andchainusingavailable enzymes in the applicationwith twosimulations. Simulation I was conducted using enzyme complex produced by Lactobacillus sp.and Rhizopus oligosporus, whilesimulation II was used enzyme complex produced by Lactobacillus sp., Rhizopus oligosporus, and Klebsiella pneumonia. Simulation I was conducted using enzyme complex produced by Lactobacillus sp. and Rhizopus oligosporus, whilesimulation II was used enzyme complex produced by Lactobacillus sp., Rhizopus oligosporus, and Klebsiella pneumoniae. A total of 58 peptides was found from the simulation I and higher than simulation I (41 peptides). The bioactive peptides by the cleavages using PeptideCuttertool were dominated with dipeptides and only three peptides were in the form of tripeptides, namely Leu-Leu-Phe(glycininG1), Val-Val-Phe(glycininG5), and Arg-His-Lys(-conglycinin-chain). Bioactive peptides with antihypertensive and antidiabetic properties were mostly found in this in silicomethod of soybean cleavage.

Familial paraganglioma may be related to mutations in succinate dehydrogenase (SDH) enzyme complex genes. Among patients with hereditary paraganglioma, SDH subunit B (SDHB) gene mutations are associated with the highest morbidity and mortality related to a higher malignancy rate. We report a family with the c.689G>A (p.Arg230His) mutation in the SDHB gene identified in two family members, a father and his daughter. While the 14-year-old daughter had no evidence of clinical disease, recurrent and later disseminated [131I]metaiodobenzylguanidine uptake-negative head and neck paraganglioma with multiple bone metastases developed in the father who underwent peptide receptor radionuclide therapy with [90Y]Y/[177Lu]Lu-dodecane tetraacetic acid octreotate (DOTATATE) at the time of the genetic diagnosis. This treatment was repeated 6 years later due to disease progression and the patient, who is currently 49 years old, remains alive and in good overall clinical condition at 8 years of follow-up after the original presentation at our unit. The growing armamentarium of imaging methods available for such patients may inform decision making regarding choice of the optimal treatment approach, potentially contributing to improved outcomes.

The influence of major nutrients’ reserves (available to plants) – nitrogen (N), phosphorus (P), potassium (K) – in the top 25-cm layer of soil on the Karelian birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti) with non-figured and figured wood via the antioxidant enzymes’ activity was investigated. The analysis of sites in area where Karelian birch trees with varying degree of figured wood intensity were growing was carried out. The cambial zone of the sample trees during active cambial growth period was studied for the activity of AOS enzyme complex (superoxide dismutase (SOD); catalase (CAT); peroxidase (POD); polyphenol oxidase (PPO)). Cellulose content and lignin content were studied. It was noticed that the availability of major nutrients in the investigated sites had influence on the degree of figured wood intensity in Karelian birch plants. Thus, non-figured and figured Karelian birch plants that grew on sites with various levels of major nutrients’ reserves differed in the AOS enzymes’ activity, which was a consequence of different xylogenesis scenarios in the studied birch forms. It was supposed, that the certain site conditions (N, P, K levels) formation could affect the degree of figured wood intensity, cellulose and lignin content. The N level and P/N ratio had the most effects under the adequate K level. So AOS enzymes’ complex activity could indicate differences in Karelian birch wood quality in sites that differ in soil fertility (N, P, K levels).

Abstract Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. Key points • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2′-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2′-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2′-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

Cellulose is considered to be one of the most underutilized biomass available on earth. These cellulosic resources, if utilized as the precursor of food, feed and biofuel can meet up the ever-increasing demands for food and energy. Since cellulase is an enzyme complex, fermentation of cellulose from agro-wastes and industrial effluents is a complex event. The physicochemical parameters are generally optimized in "one at a time" mode in fed-batch culture. But with the advent of time, statistical and mathematical modelling is used, like response surface methodology (RSM), artificial neural network (ANN), machine learning algorithm, and genetic algorithm to improve enzyme production. In bioreactor-based cellulase production, a LabVIEW-based intelligent system for monitoring bio-processing is used for the optimization of the target parameters. RSM and ANN are high-quality prediction mathematical models but ANN shows its superiority in context to the fitting of data as well as its estimation capabilities. The difference of ANN concerning RSM is its requirement of a large number of trained data. This review provides a comprehensive study of literature in context to various advanced mechanisms for optimization of cellulase production.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1S,3S)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (5), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, 14 was identified as a new selective hOAT inactivator, which demonstrated a potency 22× greater than that of 10. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of hOAT-14 and the formation of a remarkable final adduct (32') in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate 27' and elucidate the order of the two covalent bonds that formed, leading to 32'. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of 14 by hOAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for hOAT with 14 will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.

The NCW2 gene was recently described as encoding a GPI-bounded protein that assists in the re-modelling of the Saccharomyces cerevisiae cell wall (CW) and in the repair of damage caused by the polyhexamethylene biguanide (PHMB) polymer to the cell wall. Its absence produces a re-organization of the CW structure that result in resistance to lysis by glucanase. Hence, the present study aimed to extend the analysis of the Ncw2 protein (Ncw2p) to determine its physiological role in the yeast cell surface. The results showed that Ncw2p is transported to the cell surface upon O-mannosylation mediated by the Pmt1p-Pmt2p enzyme complex. It co-localises with the yeast bud scars, a region in cell surface formed by chitin deposition. Once there, Ncw2p enables correct chitin/β-glucan structuring during the exponential growth. The increase in molecular mass by hyper-mannosylation coincides with the increasing in chitin deposition, and leads to glucanase resistance. Treatment of the yeast cells with PHMB produced the same biological effects observed for the passage from exponential to stationary growth phase. This might be a possible mechanism of yeast protection against cationic biocides. In conclusion, we propose that Ncw2p takes part in the mechanism involved in the control of cell surface rigidity by aiding on the linkage between chitin and glucan layers in the modelling of the cell wall during cell growth.

Grain, in addition to starch, hemicelluloses and protein, is known to be phytic acid and its salts. It has been shown that the use of phytolytic enzymes promotes the release of phosphorus, improves the digestibility of feed nutrients and increases the meat productivity of livestock and poultry. However, the catalytic effect of phytases and their combinations with proteases on degradation of polysaccharides and protein polymers in grain raw materials, on the release of cations and anions, especially in grain wort for the production of alcohol, has been little studied. This work shows for the first time that phytolytic enzymes improve qualitative estimates, rheological properties and change the ionic composition of grain wort by not only releasing phosphates and metal cations, but also by increasing the concentration of organic salt anions. The combined action of phytolytic and proteolytic enzymes on polymers of grain raw materials provides their catalytic degradation to soluble carbohydrates, nitrogenous substances, cations and anions to produce the enriched grain wort. This work aimed to assess the degree of phytolytic and proteolytic conversion of high-molecular-weight polymers in grain wort. For grain wort, 50 g portions of wheat (Triticum sp.) or corn (Zea mays) grain flour put into Erlenmeyer flasks were added with 150 cm3 of water and incubated in a water bath. At grain batching for starch dextrinization, a thermo-stable a-amylase was added (0.6 units/g starch). For starch conversion to sugars and hydrolysis of non-starch polysaccharides in the control, we used glucoamylase (9.0 units/g starch) and xylanase (0.15 units/g raw material). Phytase (from 1.0 to 2.5 units/g raw material) and a proteolytic enzyme preparation (0.1 units/g raw material) were also added. The control was added neither with phytase, nor the proteolytic enzyme. The profiles of the main polymers of grain raw materials, the grain wort concentration, the content of reducing carbohydrates (RС) were determined according to the techno-chemical instructions for the control of alcohol production, the amine nitrogen (NH2+) concentration was measured by a method based on the ability of amino acids to form soluble copper compounds with a suspension of copper phosphate. The dynamic viscosity of the grain wort was evaluated by vibrational viscometry. The study of the ionic composition of the grain mix and wort was carried out using a PrinCE-560 series capillary electrophoresis system (PrinCE Technologies B.V., Netherlands) equipped with a conductometric detector. The optimal dosage of phytase was 1.5 units/g raw material which ensures the maximum release of ions identified by capillary electrophoresis and the effective conversion of polysaccharides, protein and phytin substances of the grain. It was found that the phytolytic enzymes contributed to a decrease in the viscosity of wheat and corn wort of more than 20 %, a 9.5-11.3 % increase in the concentration of reducing carbohydrates, and a 2.1-2.4-fold increase in the concentrations of released ions. The concentration of amino nitrogen in the wort did not change significantly. It was shown that as a result of the phytolytic action, the concentration of not only metal phosphates and cations, but also anions of organic salts, such as oxalates, malates, citrates, and succinates (in wheat wort) and oxalates, malates, citrates, and lactates (in corn wort) increased. A more significant effect of phytase for corn wort was revealed: the concentration of phosphates in the nutrient medium increased 3.9 times vs. 1.6 times for wheat wort, the levels of potassium and magnesium ions were 12 % and 22 % higher, respectively, as compared to the control in which the phytase was not used. The optimal composition of the enzyme complex is proposed which ensures the effective hydrolysis of polysaccharides, phytin and protein substances of processed raw materials. The synergistic effect of phytolytic and proteolytic enzymes enhances the catalytic hydrolysis of high molecular weight polymers of plant origin and enriches the grain wort with carbohydrates, nitrogenous substances, phosphates, and minerals in bioavailable form. Biocatalytic treatment of grain with the developed enzyme complex which, along with amylases and xylanase, contains phytase and proteases, provides a 16.8 % and 18.8 % increase in the concentration of reducing carbohydrates in wheat and corn wort, a 1.7- and 1.9-fold increase in amine nitrogen and a decrease in wort viscosity by 41.7 % and 44.7 %, respectively.

NADPH oxidase (NOX) is a membrane‐bound enzyme complex that generates reactive oxygen species (ROS). Mutations in NOX subunit genes have been implicated in the pathogenesis of inflammatory bowel disease (IBD), indicating a crucial role for ROS in regulating host immune responses. In this study, we utilize genetically deficient mice to investigate whether defects in p40phox, one subunit of NOX, impair host immune response in the intestine and aggravate disease in an infection‐based (Citrobacter rodentium) model of colitis. We show that p40phox deficiency does not increase susceptibility of mice to C. rodentium infection, as no differences in body weight loss, bacterial clearance, colonic pathology, cytokine production, or immune cell recruitment were observed between p40phox −/− and wild‐type mice. Interestingly, higher IL‐10 levels were observed in the supernatants of MLN cells and splenocytes isolated from infected p40phox‐deficient mice. Further, a higher expression level of inducible nitric oxide synthase (iNOS) was also noted in mice lacking p40phox. In contrast to wild‐type mice, p40phox −/− mice exhibited greater NO production after LPS or bacterial antigen re‐stimulation. These results suggest that p40phox −/− mice do not develop worsened colitis. While the precise mechanisms are unclear, it may involve the observed alteration in cytokine responses and enhancement in levels of iNOS and NO., We investigated the role of p40phox in Citrobacter rodentium infection. Although p40phox −/− mice did not develop worsened colitis, higher IL‐10 levels were observed in MLN cells and splenocytes isolated from infected p40phox −/− mice compared with wild‐type mice. Further, higher expression level of iNOS and greater NO production was noted in p40phox −/− lymphocytes after LPS or bacterial antigen re‐stimulation.

Aims:: The aim of this letter is to explore the influence of adding hydrogen atoms to the crystallographic structures of HIV-1 protease complexes with a series of inhibitors on the performance of radial distribution function based descriptors recently introduced in chemoinformatic studies. Background:: Quite recently the successful application of molecular descriptors based on a radial distribution function to correlate it with biologically interesting properties of a ligand – enzyme complex was demonstrated. Except its predictive power, the analysis of atoms with dominant contributions to the RDFs can be used to identify relevant atoms and interactions. Since original paper was published on dataset consisting of the X-ray structures of complexes without hydrogen atoms, we wonder weather addition of light atoms can provide us new piece of information. Objective:: The primarily objective is to create the model correlating the RDF based descriptors and physicochemical properties of the HIV-1 protease complexes with inhibitors with hydrogen atoms. Then, we will compare the performance of new model with previous one, where the hydrogen atoms were discarded. Information about interactions between the enzyme and the inhibitors will be extracted from the analysis of the RDF. Methods:: The radial distribution function descriptor weighted by the number of valence shell electrons has proven to be sensitive to the changes in the structure of the enzyme and enzyme-ligand complexes. For each structure in our data set, RDF will be calculated and using multiple linear regression method the mathematical model will be designed correlating RDF based descriptors and the physicochemical properties. Statistical analysis of the atom’s contribution to the total RDF will reveal relevant interactions. Results:: The applicability of RDF based descriptor for the correlation with pKi and EC50 values is demonstrated, while simple models containing only two or three parameters are able to explain 78 and 86 % of the variance, respectively. The models with explicitly included hydrogens are of comparable quality with the previous models without hydrogens. The analysis of the atom’s dominant contributions highlighted the importance of the hydroxyl groups of the inhibitor near the Asp25 and Asp25’ residues when it is bounded to the protease. Conclusion:: Models based on the RDF weighted by the number of valence shell electrons for correlating small number of molecular descriptors and physicocehmical properties for structures with and without hydrogens are of comparable quality and both can be used for identification of relevant functional groups and interactions. Other:: Our approach can be integrated to the next generation virtual screening methods, because is fast, reliable with high predictability potential.

Grain, in addition to starch, hemicelluloses and protein, is known to be phytic acid and its salts. It has been shown that the use of phytolytic enzymes promotes the release of phosphorus, improves the digestibility of feed nutrients and increases the meat productivity of livestock and poultry. However, the catalytic effect of phytases and their combinations with proteases on degradation of polysaccharides and protein polymers in grain raw materials, on the release of cations and anions, especially in grain wort for the production of alcohol, has been little studied. This work shows for the first time that phytolytic enzymes improve qualitative estimates, rheological properties and change the ionic composition of grain wort by not only releasing phosphates and metal cations, but also by increasing the concentration of organic salt anions. The combined action of phytolytic and proteolytic enzymes on polymers of grain raw materials provides their catalytic degradation to soluble carbohydrates, nitrogenous substances, cations and anions to produce the enriched grain wort. This work aimed to assess the degree of phytolytic and proteolytic conversion of high-molecular-weight polymers in grain wort. For grain wort, 50 g portions of wheat (Triticum sp.) or corn (Zea mays) grain flour put into Erlenmeyer flasks were added with 150 cm3 of water and incubated in a water bath. At grain batching for starch dextrinization, a thermo-stable a-amylase was added (0.6 units/g starch). For starch conversion to sugars and hydrolysis of non-starch polysaccharides in the control, we used glucoamylase (9.0 units/g starch) and xylanase (0.15 units/g raw material). Phytase (from 1.0 to 2.5 units/g raw material) and a proteolytic enzyme preparation (0.1 units/g raw material) were also added. The control was added neither with phytase, nor the proteolytic enzyme. The profiles of the main polymers of grain raw materials, the grain wort concentration, the content of reducing carbohydrates (RС) were determined according to the techno-chemical instructions for the control of alcohol production, the amine nitrogen (NH2+) concentration was measured by a method based on the ability of amino acids to form soluble copper compounds with a suspension of copper phosphate. The dynamic viscosity of the grain wort was evaluated by vibrational viscometry. The study of the ionic composition of the grain mix and wort was carried out using a PrinCE-560 series capillary electrophoresis system (PrinCE Technologies B.V., Netherlands) equipped with a conductometric detector. The optimal dosage of phytase was 1.5 units/g raw material which ensures the maximum release of ions identified by capillary electrophoresis and the effective conversion of polysaccharides, protein and phytin substances of the grain. It was found that the phytolytic enzymes contributed to a decrease in the viscosity of wheat and corn wort of more than 20 %, a 9.5-11.3 % increase in the concentration of reducing carbohydrates, and a 2.1-2.4-fold increase in the concentrations of released ions. The concentration of amino nitrogen in the wort did not change significantly. It was shown that as a result of the phytolytic action, the concentration of not only metal phosphates and cations, but also anions of organic salts, such as oxalates, malates, citrates, and succinates (in wheat wort) and oxalates, malates, citrates, and lactates (in corn wort) increased. A more significant effect of phytase for corn wort was revealed: the concentration of phosphates in the nutrient medium increased 3.9 times vs. 1.6 times for wheat wort, the levels of potassium and magnesium ions were 12 % and 22 % higher, respectively, as compared to the control in which the phytase was not used. The optimal composition of the enzyme complex is proposed which ensures the effective hydrolysis of polysaccharides, phytin and protein substances of processed raw materials. The synergistic effect of phytolytic and proteolytic enzymes enhances the catalytic hydrolysis of high molecular weight polymers of plant origin and enriches the grain wort with carbohydrates, nitrogenous substances, phosphates, and minerals in bioavailable form. Biocatalytic treatment of grain with the developed enzyme complex which, along with amylases and xylanase, contains phytase and proteases, provides a 16.8 % and 18.8 % increase in the concentration of reducing carbohydrates in wheat and corn wort, a 1.7- and 1.9-fold increase in amine nitrogen and a decrease in wort viscosity by 41.7 % and 44.7 %, respectively.

The aim of this work was to study the composition and functions of intracellular and extracellular proteases of the production Vibrio cholerae strains 569B serovar Inaba and M41 serovar Ogawa using zymographic and proteomic analysis.Materials and methods. Samples of intracellular proteases were obtained from cell lysates by ultrasonication of bacterial cells in a 9 M urea solution. The extracellular protease fraction was precipitated from the culture liquid by adding 50 % trichloroacetic acid to a final concentration of 10 % and incubating on ice. Lyophilized preparations of proteinase K and proteovibrin enzyme complex were used as a control of proteolytic activity. Proteases were detected by substrate gel electrophoresis in 12.5 % polyacrylamide gel impregnated with 0.1 % gelatin, followed by identification of the composition of protein fractions of lysates and exoproteins of both strains using molecular mass spectrometric scanning.Results and discussion. A comparative study of the production strains of V. cholerae 569B serovar Inaba and M41 serovar Ogawa using zymographic and proteomic analysis showed that the greatest enzymatic activity was detected in the fraction of extracellular proteases sample of V. cholerae M41 strain, where five major and four minor zones of gelatin hydrolysis were identified, and high-intensity zones with MW 20–23 and 37–40 kDa were also found in the preparation of proteovibrin isolated from the culture fluid of that strain. As a result of proteomic analysis of the studied strains, 66 enzymes of V. cholerae with different functional activity were reliably identified, among which 15 enzymes had protease activity. The high information content of the complex of modern methods provided for the possibility of identifying qualitative and quantitative differences in the composition of intracellular and extracellular proteases in production strains of V. cholerae, which offers an effective means of screening inter-strain differences in the protease spectrum in production strains.

The current research focused on the isolation of a yeast strain possessing an enzyme complex for use in baking high quality, high cassava-wheat composite bread. Yeast isolates were obtained from so...

Selitrectinib (BAY2731954; LOXO-195) is a promising oral tropomyosin receptor kinase (TRK) inhibitor currently in phase I/II clinical trials for the treatment of histology-agnostic cancers positive for TRK fusions. With therapeutic resistance eventually developing with first-generation TRK inhibitors, selitrectinib was designed to overcome resistance mediated by acquired kinase domain mutations. Using genetically modified mouse models and pharmacological inhibitors, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing CYP3A enzyme complex in selitrectinib pharmacokinetics. In vitro, selitrectinib was markedly transported by mouse Abcg2 and human ABCB1, and modestly by human ABCG2. Following oral administration at 10 mg/kg, selitrectinib brain-to-plasma ratios were increased in Abcb1a/1b−/− (twofold) and Abcb1a/1b;Abcg2−/− (5.8-fold) compared with wild-type mice, but not in single Abcg2−/− mice. Testis distribution showed similar results. mAbcb1a/1b and mAbcg2 each restricted the plasma exposure of selitrectinib: With both systems absent oral availability increased by 1.7-fold. Oral administration of the ABCB1/ABCG2 inhibitor elacridar boosted plasma exposure and brain accumulation in wild-type mice to the same levels as seen in Abcb1a/1b;Abcg2−/− mice. In Cyp3a−/− mice, plasma exposure of selitrectinib over 4 hours was increased by 1.4-fold and subsequently reduced by 2.3-fold upon transgenic overexpression of human CYP3A4 in liver and intestine. The relative tissue distribution of selitrectinib remained unaltered. Thus, selitrectinib brain accumulation and oral availability are substantially restricted by ABCB1 and ABCG2, and this can be reversed by pharmacological inhibition. Moreover, oral availability of selitrectinib is limited by CYP3A activity. These insights may be useful to optimize the clinical application of selitrectinib.

Purpose Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic disorder characterized by a deficiency of the sucrase-isomaltase (SI) enzyme complex within the brush border membrane of the small intestine. Mutations in the SI gene result in abnormal synthesis and/or incorrect transport of the SI enzyme. Patients with CSID generally have reduced sucrase activity, but levels of isomaltase activity range from absent to almost normal. This study sought to better understand the experience of patients with CSID prior to, during, and after their diagnosis and its subsequent treatment with sacrosidase. Methods This was a cross-sectional interview study conducted in conjunction with a longitudinal, observational study of US patients prescribed and taking sacrosidase for at least three consecutive months as treatment for CSID. The observational study included both children and adults. Results This qualitative interview study explored the experiences of 43 adult and pediatric patients (n = 8 adults and n = 35 children/adolescents) with CSID pre-, during, and post-diagnosis. Findings suggest that a CSID diagnosis is particularly problematic given the disparate range of more commonly understood gastrointestinal (GI) disorders. After diagnosis and treatment with sacrosidase, participants reported considerable improvement in symptoms and health-related quality of life (HRQL), yet symptoms persist that continue to affect daily life, indicating areas of potential unmet need. Conclusion Educating clinicians about CSID may help improve the overall diagnosis experience. As this research is the first of its kind in CSID, additional research, qualitative and quantitative, will be important to furthering the understanding of HRQL impact and unmet need experienced by this population and identifying ways to best meet those needs.

With the progress of human society, the consumption of energy materials is increasing in particularly the fossil fuels, which accounts for 84.7% of the total global consumption in 2018. Thus, excessive exploitation of fossil fuels not only brings about an increasingly serious energy crisis, but also leads to a series of environmental problems, such as the air quality deterioration and soil acidification, which are not conducive to the sustainable development of society. Since the 21st Annual Conference of the China Association for Science and Technology in 2019, “renewable synthetic fuels” has become one of the focuses of attention, and it also puts forward higher requirements for the research and development of renewable energy. About 60 years ago, many plants, insects, microorganisms and microalgae were found to metabolize hydrocarbons. In 2010, Schirmer’s groups recognized the alkane biosynthesis pathways of marine chlorobacteria, which aroused great interest in hydrocarbon biosynthesis. Recently, more proteases to catalyze the synthesis of hydrocarbons have been successively discovered, such as aldehyde deformylase (ADO, CYP4G and CER), fatty acid decarboxylase (UndA, UndB and OleT), polyketone synthase (Ols) and thiolytic enzyme complex system (OleABCD). In the past decades, many studies have been conducted to explore the biosynthesis of hydrocarbon, and useful information regarding the reaction mechanism has been obtained, including the kinetic rate constants and the crystal structures of the enzymes. In addition, on the basis of crystal structure, quantum chemistry method (QM) and molecular dynamics simulations (MD) were also used to explore the reaction mechanism of fatty acid decarboxylation. Since most enzymes involved in the hydrocarbon biosynthesis are iron-containing enzymes, which employ the Fe-coordinate dioxygen to trigger the reaction. In general, the whole reaction cycle contains the binding of substrates and dioxygen, and decarboxylation usually involves the hydrogen atom abstraction and breaking of C−C bond. During the reaction, complex electron transfer takes place among the Fe center, O2 and substrate, and many factors were found to affect the catalytic activity. According to the previous experimental and theoretical studies, different enzymes show different activity and substrate scope. In general, the catalytic efficiency is still very low for most enzymes, and thus, understanding the mechanism of these catalytic reactions is undoubtedly an important basis for engineering optimization and all applications, and can also provide ideas for the design and synthesis of biomimetic catalysts. In this paper, we review the research progress of five representative iron enzymes that catalyze the biosynthesis of aliphatic hydrocarbon, including OleT, UndA, UndB, ADO and CYP4G, and discuss the future development and application of enzyme catalysis in renewable synthetic fuels. This paper may offer some ideas and guidelines for sustainable energy supply in the future.

Pyridoxal-5'-phosphate (PLP), the biologically active form of vitamin B6, serves as a cofactor for many enzymes. The Gram-positive model bacterium Bacillus subtilis synthesizes PLP via the PdxST enzyme complex, consisting of the PdxT glutaminase and the PdxS PLP synthase subunits, respectively. PdxT converts glutamine to glutamate and ammonia of which the latter is channelled to PdxS. At high extracellular ammonium concentrations, the PdxS PLP synthase subunit does not depend on PdxT. Here, we assessed the potential of a B. subtilis ΔpdxT mutant to adapt to PLP limitation at the genome level. The majority of ΔpdxT suppressors had amplified a genomic region containing the pdxS gene. We also identified mutants having acquired as yet undescribed mutations in ammonium assimilation genes, indicating that the overproduction of PdxS and the NrgA ammonium transporter partially relieve vitamin B6 limitation in a ΔpdxT mutant when extracellular ammonium is scarce. Furthermore, we found that PdxS positively affects complex colony formation in B. subtilis. The catalytic mechanism of the PdxS PLP synthase subunit could be the reason for the limited evolution of the enzyme and why we could not identify a PdxS variant producing PLP independently of PdxT at low ammonium concentrations.