Amount Of Enzyme Research Articles

Influence of Cotton Bleaching Methods on the Parameters of Generated Textile Industrial Wastewater

The environmental impact of industrial wastewater after bleaching requires special attention to prevent the pollution of aquatic ecosystems. The aim of this study was to compare the effluent parameters obtained after using a traditional bleaching method and an alternative enzymatic bleaching method in order to assess their possible environmental impacts. In the classical bleaching method, NaOH and H2O2 were used, and in the alternative method, pectinase enzyme, H2O2, and sodium carbonate were used as the bleaching agents. The bleaching process was designed by determining the optimum amount of pectinase enzyme, the optimum temperature, and the optimum amount of peroxide. The whiteness of the treated cotton yarns was also evaluated. The different bleaching processes were analyzed and evaluated in terms of the wastewater discharged, the pH, chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS), and conductivity. These data show that the use of the pectinase enzyme allows us to obtain much lower COD and TSS values compared to those obtained using traditional bleaching. At the same time, the use of the proposed enzymatic bleaching method reduces the environmental impact since lower concentrations of pollutants are obtained in the effluent. Therefore, the pretreatment of wastewater may require the selection of a simpler and more environmentally friendly technology, such as enzymatic bleaching.

Assessment of the Roles of Magnesium and Zinc in Clinical Disorders.

The ability and facility of magnesium (Mg2+) and zinc (Zn2+) to interact with phosphate ions confer them the characteristics of essential trace elements. Trace elements are extremely necessary for the basic nucleic acid chemistry of cells of all known living organisms. More than 300 enzymes require zinc and magnesium ions for their catalytic actions, including all the enzymes involved in the synthesis of ATP. In addition, enzymes such as isomerases, oxidoreductases, lyases, transferases, ligases and hydrolases that use other nucleotides to synthesize DNA and RNA require magnesium and zinc. These nucleotides may trigger oxidative damage or important changes against free radicals. In the same way, nucleotides may play an important role in the pathophysiology of degenerative diseases, including in some clinical disorders, where vascular risk factors, oxidative stress and inflammation work to destabilize the patients` homeostatic equilibrium. Indeed, reduced levels of zinc and magnesium may lead to inadequate amount of antioxidant enzymes, and thus, acts as an important contributing factor for the induction of oxidative stress leading to cellular or tissue dysfunction. Hence, the development of zinc or magnesium enzyme inhibitors could be a novel opportunity for the treatment of some human disorders. Therefore, the objective of the present work was to assess the clinical benefits of zinc and magnesium in human health and their effects in some clinical disorders.

PSIV-20 An in Vitro Digestion Procedure Using Daisyii Incubator Can Replace Conventional in Vitro Digestion Procedures Using Flasks for Estimating Ileal Digestibility of Feed Ingredients for Pigs

Abstract In vitro procedures using flasks have been used for simulating nutrient utilization by pigs. However, these procedures are time-consuming and laborious and require a number of flasks and filter crucibles. The objective of this study was to develop an in vitro procedure using DaisyII incubator for simultaneous analysis of multiple samples. In a conventional in vitro procedure, 10 mg of pepsin in step 1 and 50 mg of pancreatin in step 2 are used for each flask containing 1 g of feed sample. In a preliminary experiment, 24 samples each in R55 bags were digested in a jar of DaisyII incubator with 24 times the quantity of buffer solution and enzymes of the conventional flask procedure. In the preliminary experiment, greater crude protein (CP) digestibility values were observed in the DaisyII incubator procedure compared with the conventional flask procedure. To determine optimum enzyme doses in the in vitro ileal disappearance (IVIDDaisy) procedure using the DaisyII incubator, 3 doses of pepsin (10, 7.5, and 5 mg/sample in step 1) and 3 doses of pancreatin (50, 37.5, and 25 mg/sample in step 2) were tested in a 3 × 3 factorial arrangement for 11 test ingredients. The quantity of buffer solution and enzymes in a jar was increased proportionally with the number of samples. The 11 test ingredients were barley, copra meal, corn, corn distillers dried grains with solubles, corn gluten feed, corn gluten meal, palm kernel meal, rice bran, soybean hulls, soybean meal, and wheat bran. The in vitro ileal disappearance (IVIDflask) of CP in the 11 ingredients was determined using the conventional flask procedure. The sum of square of deviation between IVIDflask of CP and IVIDDaisy of CP was the least when 5 mg pepsin/sample in step 1 and 37.5 mg/sample in step 2 were used (IVIDflask of CP, % = 14.4 + 0.81 × IVIDDaisy of CP, %; r2 = 0.748). On the other hand, the determination coefficient was the greatest when 10 mg pepsin/sample in step 1 and 50 mg/sample in step 2 were used (IVIDflask of CP, % = 2.5 + 0.93 × IVIDDaisy of CP, %; r2 = 0.878). In conclusion, the in vitro procedure using a DaisyII incubator can be used for estimating ileal digestibility of protein in multiple samples simultaneously for pigs.

Development and Identification of Novel α-Glucosidase Inhibitory Peptides from Mulberry Leaves.

The mulberry leaf is a botanical resource that possesses a substantial quantity of protein. In this study, alcalase hydrolysis conditions of mulberry leaf protein were optimized using the response surface method. The results showed that the optimum conditions were as follows: substrate protein concentration was 0.5% (w/v), enzymatic hydrolysis temperature was 53.0 °C, enzymatic hydrolysis time was 4.7 h, enzyme amount was 17,800 U/g, and pH was 10.5. Then mulberry leaf peptides were separated by ultrafiltration according to molecular weight. Peptides (<3 kDa) were screened and subsequently identified using LC-MS/MS after the evaluation of α-glucosidase inhibition across various fractions. Three novel potential bioactive peptides RWPFFAFM (1101.32 Da), AAGRLPGY (803.91 Da), and VVRDFHNA (957.04 Da) with the lowest average docking energy were screened for molecular dynamics simulation to examine their binding stability with enzymes in a 37 °C simulated human environment. Finally, they were prepared by solid phase synthesis for in vitro verification. The former two peptides exhibited better IC50 values (1.299 mM and 1.319 mM, respectively). These results suggest that the α-glucosidase inhibitory peptides from mulberry leaf protein are potential functional foods or drugs for diabetes treatment, but further in vivo studies are needed to identify the bioavailability and toxicity.

A Simple Validated Method for the Estimation of Pepsin Activity in Microtiter Array for the INFOGEST Protocol.

The INFOGEST protocol has been widely used as a static in-vitro simulation of gastrointestinal food digestion for bioaccessibility assessments on bioactive compounds. The standardization of the activity of several enzymes, such as pepsin, via UV-spectrophotometry of digested hemoglobin at 280 nm is a key step in the protocol. Standardization is a crucial stage since it is necessary to determine the quantity of enzyme to be added to the sample for digestion. However, this method is yet to be analytically validated; it requires quartz cuvettes and large volumes of samples and is time-consuming. Thus, we reviewed and adapted a well-known colorimetric method in microplates array by using the Folin-Ciocalteu reagent, and this study is the first to report for miniaturization of this method, the advantages of which include its automation, ease of use, the low volume of samples required, the minimal use of reagents, and speed. This method was compared to the traditional UV method, and the comparison results show no statistical difference between the inter day means for each group (p > 0.05). The proposed method was validated, showing high reproducibility (8% as inter-day CV) and statistically comparable results with the traditional UV spectrophotometric method.

Emmer starch isolation by alkaline protease digestion of wet-milled seeds

In today's dynamic world, it is necessary to explore more alternative options for starch isolation and develop technologies to obtain better quality products. The presence of impurities in the composition of starch is an indicator that affects its properties. Starch was isolated from whole emmer seeds. The present study aims to compare the protein content of isolated emmer starch obtained by a standard alkaline method and a combined alkaline with an enzymatic method. The conducted method for the isolation of starch from emmer was with a duration of the alkaline treatment of 24 and 48 hours. The results were compared with the results obtained from the combination of alkaline (24 hours) and enzyme (with an amount of enzyme 0.05 - 2.2% for 1 - 8 h) treatment. The results showed that the long alkaline treatment could be replaced by a shorter one with 0.05% enzyme for 1 hThe lowest values of protein in the composition of starch were reported after treatment with 1.1% multienzyme preparation for 8 h.

Assessment of steroid enzymes action in children and adolescents with obesity

Rising prevalence of obesity has become an important impulse to investigate basic mechanisms involved in regulating the energy balance. It is widely accepted that steroids are potent factors affecting glucose, fat, and protein metabolism. Our study was aimed to analyze differences in the total amount of selected enzymes implicated in steroid metabolism in a group of children suffering from obesity and those with normal weight, further subdivided according to sex and pubertal stage. Data were obtained from 187 Caucasian children and adolescents, including 113 patients (63 girls, 50 boys) with obesity and 74 (34 girls, 40 boys) normal weight volunteers. Standard clinical examinations were performed in both groups. To evaluate the impact of puberty, preadolescent children and those with advanced puberty were assessed separately. Urine steroid excretion profiles were analyzed using gas chromatography/mass spectrometry method. Children with obesity revealed several changes in in the total amount of steroid enzymes as assessed by the relevant metabolite proportions, compared to their norm weight peers. Girls showed a significant increase in the activity of 11βHSD1, while boys demonstrated a relevant elevation in 20αHSD action. Regardless of sex, children with obesity showed an increase in the activity of 5β-reductase + 3αHSD complex and a decrease in the involvement of 11βOH-lase. The effect is attenuated when consider pre- and pubertal subgroups. We hypothesize that changes in the activity levels of selected enzymes may be a compensatory mechanism to limit the glucocorticoid exposure of key target tissues as well as to improve metabolic control and reduce long-term complications of obesity.

Production of Copper Nanoparticles by Aspergillus niger and Evaluation of their Biomedical Applications

Nanotechnology is a well advanced area and different types of nanoparticles are now produced. The most revolutionary topic of the twenty-first century is nanotechnology. Recently, metallic nanoparticles (NPs) have gained a lot of attention. In this study a straightforward, safe, and economical method is used for synthesizing copper nanoparticles (CuNPs) by using cell free filtrate of Aspergillus niger. Fungi secretes enormous amount of enzymes which act as reducing as well stabilizing agents. Synthesized CuNPs were characterized by different techniques. Ultraviolet (UV)-visible spectrophotometer showed a characteristic peak at 380nm, Dynamic Light Scattering (DLS) analysis indicated that CuNPs have average size of 570nm with 0.480 PDI. SEM (Scanning Electron Microscopy) revealed CuNPs are of cubic shape. As per FTIR (Fourier Transform Infrared Analysis) two functional groups i.e., -O-H- and C=C- on the surface of CuNPs. Genetic analysis of Aspergillus niger revealed the presence of nitrate reductase enzyme that may involve in reduction of metal into NPs. Furthermore, CuNPs showed significant antibacterial action against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Klebsiella pneumoniae with 17mm, 20mm, 17mm and 20mm respectively. CuNPs also showed antioxidant potential with 59.41µg/ml.&#x0D; Fungi secretes valuable enzymes with inherent ability to reduce metal ions into NPs. This reduced the cost of NPs synthesis and also synthesized NPs exhibit greater biomedical applications.

Enzyme immobilized on magnetic fluorescent bifunctional nanoparticles for α-glucosidase inhibitors virtual screening from Agrimonia pilosa Ledeb extracts accompanied with molecular modeling

Agrimonia pilosa Ledeb (APL) is a significant source of inhibitors for α-glucosidase, which is an essential target enzyme for the treatment of type 2 diabetes, cancer and acquired immune deficiency syndrome. Ligand fishing is a suitable approach for the highly selective screening of bioactive substances in complex mixtures. Yet it is unable to conduct biomedical imaging screening, which is crucial for real-time identification. In this case, a bioanalytical platform combining magnetic fluorescent ligand fishing and in-situ imaging technique was established for the screening and identification of α-glucosidase inhibitors (AGIs) from APL crude extract, utilizing α-glucosidase coated CuInS2/ZnS-Fe3O4@SiO2 (AG-CIZSFS) nanocomposites as extracting material and fluorescent tracer. The AG-CIZSFS nanocomposites prepared through solvothermal and crosslinking methods displayed fast magnetic separation, excellent fluorescence performance and high enzyme activity. The tolerance of immobilized enzyme to temperature and pH was stronger than that of free enzyme. Prior to proof-of-concept with APL crude extract, a number essential parameters (glutaraldehyde concentration, immobilized time, enzyme amount, reaction solution pH, incubation temperature, incubation time, percentage of methanol in eluen, elution times and eluent volume) were optimized using an artificial test mixture. The fished ligands were identified by UPLC-MS/MS and their biological activities were preliminarily evaluated by real-time cellular morphological imaging of human colon carcinoma (HCT-116) cells based on confocal laser scanning microscope (CLSM). Their α-glucosidase inhibitory activities were further verified and studied by classical pNPG method and molecular docking. The isolated compounds exhibited significant α-glucosidase inhibitory activities with a IC50 value of 11.57 µg·mL−1. Six potential AGIs including tribuloside, ivorengenin A, tormentic acid, 1β, 2β, 3β, 19α-Tetra hydroxyurs-12-en-28-oic acid, corosolic acid and pomolic acid were ultimately screened out and identified from APL crude extracts. The proposed approach, which combined highly specific screening with in-situ visual imaging, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicine (TCM).

The Exogenous Application of 24-Epibrassinolide (24-EBL) Increases the Cd and Pb Resilience in Zea mays (L.) by Regulating the Growth and Physiological Mechanism.

Heavy metals (HMs) at a concentration above the thresholdlevel act as environmental pollutants and very often threaten theagricultural productivity globally. Finding affordable and environmentally sustainable deliverables to address this issue is therefore a top focus. Phytohormones alleviate the HMs-induced toxicity and positively influence the plant growth. Considering the importance of phytohormones, the present study aimed to assess the effect of 24-epibrassinolide (24-EBL; 10 µM) as seed soaking treatment on growth performance of Zea mays (L.) contaminated separately with increasing concentrations (50-400 mg.kg-1) of lead (Pb) and cadmium (Cd). With increasing metal concentrations, growth and plant biometric criteria were reduced. For instance, Cd at 400 mg.kg-1 soil reduced the germination efficiency (56%), root (77%) and shoot (69%) dry weight, total chlorophyll (64%), and carotenoid content (45%). Contrarily, both HMs caused increase in stress biomarkers and antioxidant enzymes in seedling. However, exogenous administration of 24-EBL significantly enhanced the growth attributes, photosynthetic pigments, proline, MDA, and antioxidant enzyme activity while reducing the harmful effects of HMs stress on Z. mays. For instance, 24-EBL (10 µM) improved the germination percentage, root biomass, chl a, chl b, total chlorophyll, and carotenoid content by 16, 21, 17, 34, 18, and 15%, respectively, in 50 mg.Pb.kg-1 soil-treated Z. mays plants. Furthermore, the amounts of proline, MDA, and antioxidant enzymes in foliage of Z. mays were interestingly and dramatically lowered by 24-EBL application. Uptake of metals in plant organs was significantly reduced when 24-EBL was applied to Pb- and Cd-treated Z. mays. The recent findings help us better understand how 24-EBL regulates growth and development of Z. mays as well as how it boosts HMs' resilience, which could increase the possibility of employing 24-EBL to increase Z. mays productivity. Thus, the present findings confirmed the potentiality of pre-soaking the seed in 24-EBL solution that neutralizes the toxic effects of heavy metals in Z. mays plants. Therefore, it is suggested that applying phytohormones including 24-EBL in removal of heavy metal stress in plants is the best possible solution in sustainable agriculture.

Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli

Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA β-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.

Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications.

In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes' performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used. Here, we pave the way for a standardized enzymatic PET hydrolysis protocol using reaction conditions relevant for larger scale hydrolysis and apply these parameters to four recently reported PET hydrolases (LCCICCG, FAST-PETase, HotPETase, and PES-H1L92F/Q94Y). We show that FAST-PETase and HotPETase have intrinsic limitations that may not permit their application on larger reaction scales, mainly due to their relatively low depolymerization rates. With 80% PET depolymerization, PES-H1L92F/Q94Y may be a suitable candidate for industrial reaction scales upon further rounds of enzyme evolution. LCCICCG outperforms the other enzymes, converting 98% of PET into the monomeric products terephthalic acid (TPA) and ethylene glycol (EG) in 24 h. In addition, we optimized the reaction conditions of LCCICCG toward economic viability, reducing the required amount of enzyme by a factor of 3 and the temperature of the reaction from 72 to 68 °C. We anticipate our findings to advance enzymatic PET hydrolysis toward a coherent assessment of the enzymes and materialize feasibility at larger reaction scales.

Pharmacodynamics, safety, tolerability and pharmacokinetics of a single oral dose of an engineered phenylalanine ammonia-lyase in patients with phenylketonuria

The cornerstone treatment of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) is a lifelong low-protein diet with phenylalanine (Phe) free L-amino acid supplements. However, the PKU diet has significant shortcomings, and there is a clinically unmet need for new therapeutics to improve patient outcomes. CDX-6114 is a modified phenylalanine ammonia-lyase (PAL) enzyme obtained by a mutation in the Anabaena variabilis PAL sequence. CodeEvolver® protein engineering technology has been applied to improve the degradation resistance of the enzyme. In our first phase I trial, 19 patients were given a single oral dose of CDX-6114 at 7.5 g, 2.5 g, 0.7 g, or placebo in a cross-over design. After an overnight fast, patients received a standardised breakfast of 20 g of protein, thus exceeding the dietary recommendations for a single meal in patients with PKU. Plasma levels of Phe and cinnamic acid (CA) were measured over a 5-h period following CDX-6114 dosing. During the development of CDX-6114, a stability assessment using reverse-phase high-performance liquid chromatography (HPLC) assay revealed two peaks. The second peak was identified as CA. It was not previously known that as part of the mechanism of action, the CA remained associated with the protein following the conversion of Phe. Thus, recalculating the historical PAL enzyme amounts in CDX-6114 bulk substance was necessary. An updated extinction coefficient was achieved by applying a correction factor of 0.771 to previously reported doses. Postprandial plasma levels of Phe increased in all dose cohorts over time between 10% and 30% from baseline, although the actual peak of Phe levels was not achieved within the 5-h observation. When accounting for the interquartile ranges, these concentrations were similar to the placebo. As plasma levels of Phe were no longer a reliable marker for pharmacodynamics, the consistently detectable amount of CA seen in all patients who received CDX-6114 provided proof of the enzymatic activity of CDX-6114 in metabolising gastrointestinal Phe. Peak levels of CA were seen shortly after CDX-6114 intake, with a rapid decline, and remained low compared with the plasma Phe levels. This pattern indicates a short half-life, possibly due to the liquid formulation or the inability to withstand the lower pH in the human stomach compared with animal models in earlier studies. This was the first trial in patients with PKU to establish the safety and tolerability of CDX-6114. A single dose of CDX-6114 was safe and well tolerated, with no serious adverse events or presence of anti-drug antibodies detected. Efficacy will be explored in future trials using an optimised formulation.

In vitro screening of crude urease extract from native Australian plants and weeds for bio-cementation of sands

Urease enzymes from plants are directly utilised in enzyme-induced calcite precipitation (EICP) to catalyse calcium carbonate (CaCO3) precipitation between soil particles to improve their strength and stiffness. EICP does not require microbial culture and may be applicable for finer soils with their smaller size. However, most studies on EICP utilise purified urease enzymes, which are often rare, expensive and limited to food-grade beans or seeds, making the technique less cost-effective. To find alternative and cheaper sources, crude urease extracts from diverse plant species, particularly weeds and Australian native plants, were obtained, identified and characterised using a series of in vitro experiments. All selected plant species contain a considerable amount of urease enzyme, exhibiting different urease activity and calcium carbonate precipitation. EICP treatment by different crude extracts showed different levels of hydraulic conductivity, unconfined compressive strength (UCS), wind and raindrop erosion resistance. However, the crude extract from an Australian weed, matured paddy melon seeds (M-PMS), had the highest specific activity of 8997 U/mg and was further used for EICP treatment of six different soil types. The UCS of treated soils was influenced by the soil types. For similar calcium carbonate content, the strength and stiffness of the EICP-treated specimens were significantly influenced by the confining stress and degree of saturation in undrained triaxial conditions. Also, the mass loss, strength and average calcium carbonate decreased with increasing cycles of wetting–drying, freezing–thawing and elevated temperature. When compared with purified urease enzyme, M-PMS produced comparable strength and durability resistance in the treated samples.

Pichia pastoris growth—coupled heme biosynthesis analysis using metabolic modelling

Soy leghemoglobin is one of the most important and key ingredients in plant-based meat substitutes that can imitate the colour and flavour of the meat. To improve the high-yield production of leghemoglobin protein and its main component—heme in the yeast Pichia pastoris, glycerol and methanol cultivation conditions were studied. Additionally, in-silico metabolic modelling analysis of growth-coupled enzyme quantity, suggests metabolic gene up/down-regulation strategies for heme production. First, cultivations and metabolic modelling analysis of P. pastoris were performed on glycerol and methanol in different growth media. Glycerol cultivation uptake and production rates can be increased by 50% according to metabolic modelling results, but methanol cultivation—is near the theoretical maximum. Growth-coupled metabolic optimisation results revealed the best feasible upregulation (33 reactions) (1.47% of total reactions) and 66 downregulation/deletion (2.98% of total) reaction suggestions. Finally, we describe reaction regulation suggestions with the highest potential to increase heme production yields.

Optimized Apparatus Design for Continuous Aqueous Two-Phase Flotation (ATPF)

Aqueous two-phase flotation (ATPF) is an integrative downstream method for separating and concentrating biomolecules from crude biosuspensions such as fermentation broth. Its continuous operation not only increases throughput, but also makes it recommended for continuous upstreams. In this work, an optimized apparatus design in the form of a horizontal flotation tank is presented. The geometry of the new apparatus for continuous ATPF is optimized using flow simulations so that the two aqueous phases can be pumped uniformly through the flotation tank in co-current or counter-current flow. The implementation of a conductivity measurement enables to characterize the phase mixing effect induced by the rising gas bubbles introduced through three different gassing units. ATPF experiments with the model enzyme phospholipase A2 show that there are favorable combinations of gas flow rates to increase the flotation rate and reduce the back-diffusion at the outlet of the apparatus. This allows high phase exchange rates up to 2/h and hence increases the amount of enzymes that can be recovered per time. Co-current operation of continuous ATPF increased separation efficiency about 14% up to E = 74% compared to counter-current operation.

Obtaining protein hydrolysate from secondary meat raw materials for food enrichment

Diseases of the musculoskeletal system are the most common in the elderly. Collagen hydrolysates obtained from animal raw materials are widely used to prevent and reduce the risk of such diseases. The effectiveness of collagen hydrolysates as an independent product and as part of food products has been confirmed. A study of the technological process of obtaining hydrolysates from inexpensive raw materials, such as wool by-products, using enzymatic hydrolysis. The main purpose of the study was to identify factors affecting the accumulation of hydrolysis products. To achieve this goal, the main parameters of hydrolysis were optimized, including the duration of the process, the temperature regime and the amount of enzyme added in the process. The resulting protein hydrolysate contains a high concentration of glycine – 27,160 %, 13,317 %, 13,216 %, valine and proline showed almost the same high level – 16 %, 10 %, 8,8 %, alanin – 10,494 %, 7,022 %, 6,681 %, arginine – 6,173%, 3,874%, 6,021% depending on raw materials. The results of the studies showed that the optimal parameters for obtaining hydrolysate were achieved using an enzyme preparation in an amount of 5% of the total mass, temperature 45 ° C, duration of hydrolysis 24 hours. The hydrolysate obtained as a result of this process can serve as the basis for the production of functional products that are intended for the elderly.

Endogenous chitinase might lead to differences in growth performance and intestinal health of piglets fed different levels of black soldier fly larva meal.

This study aimed to investigate the effects of different levels of black soldier fly (BSF) replacing soybean meal (SBM) in diets on the performance and health condition of piglets. A total of 180 weaned piglets were allocated into 5 treatments: BSF0 (corn-soybean meal basal diet), BSF25 (BSF replacing 25% SBM), BSF50 (BSF replacing 50% SBM), BSF75 (BSF replacing 75% SBM) and BSF100 (BSF replacing 100% SBM). During the whole period, in comparison with BSF0, average daily gain (ADG) and average daily feed intake increased in the BSF25 and BSF50 groups, whereas ADG decreased in the BSF75 and BSF100 groups (P<0.05). The result of quadratic fitting curve showed that piglets exhibited the highest ADG when BSF replaced around 20% SBM. Compared with BSF0, organic matter and dry matter digestibility improved in the BSF25 group, whereas ether extract digestibility decreased in the BSF100 group (P<0.05). In comparison with BSF0, piglets from the BSF25 group showed a higher duodenal ratio of villus height to crypt depth, increased jejunal sucrase activity, serum neuropeptide Y and ghrelin levels, elevated ileal immunoglobulin (Ig) A, IgG and IgM contents and a lower leptin level, and piglets from the BSF100 group exhibited an increased relative weight of kidney (P<0.05). However, no significant differences were observed in the expression level of tight junction proteins and chitin-degrading enzyme. Additionally, compared with BSF0, the abundance of short chain fatty acid producing bacteria such as Ruminococcaceae, Faecalibacterium and Butyricicoccus increased, and potential pathogenic bacteria decreased in piglets from the BSF25 group, whereas piglets from the BSF100 group had a greater abundance of harmful bacteria. In conclusion, BSF replacing 25% SBM in diets could improve digestive parameters, immune function and intestinal microbiota, and thus improved growth performance of piglets. However, BSF replacing 100% SBM showed an adverse effect on piglet performance, and the reason might be related to the limited amount of chitin-degrading enzyme.

Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells.

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

Effects of Phosphorus-dissolving Dark Septate Endophytes on the Growth of Blueberry.

Dark septate endophytes (DSEs) are widely distributed and improve plant growth. DSEs secrete large amounts of enzymes to mineralize insoluble phosphorus in soil and convert it into soluble phosphorus, promoting plant uptake of phosphorus. However, the effects of DSEs with phosphate-solubilizing ability on host plants need further study. In this study, phosphorus-dissolving DSEs were screened for growth-promoting effects. We isolated, identified and characterized three DSE species (Thozetella neonivea, Pezicula ericae and Hyaloscyphaceae sp.) showing phosphate-solubilizing ability. The impact of single, dual or triple inoculation of DSEs on blueberry plant characteristics was studied. Their effects on colonization intensity, seedling biomass, nutrients in plants and soil, and activities of plant resistance enzymes and soil enzymes were markedly upregulated relative to the control (P < 0.05). The available phosphorus and acid phosphatase levels in different combinations were significantly increased. These findings indicate that the application of the three DSEs may be valuable in facilitating the cultivation of blueberry with a higher biomass and improved plant quality.