Enzyme Fraction Research Articles

Biocontrol of fungal pathogens and growth promotion in the Korean fir (Abies koreana E.H.Wilson) seedling using Bacillus velezensis CE 100

The restoration of the endangered Korean fir (Abies koreana E.H.Wilson) forests requires mass production of high-quality and well-developed seedlings for outplanting. However, phytopathogenic fungal infections lower the survival rate and growth vigor of A. koreana seedlings, frustrating the re-afforestation efforts. This study isolated and identified the phytopathogenic fungi causing seedling blight and wilt diseases in A. koreana seedlings and investigated the potential of Bacillus velezensis CE 100 in controlling the fungal pathogens and promoting seedling growth. Phomopsis mali and Fusarium oxysporum were confirmed as the causal agents of blight and wilt diseases, respectively, and both fungi reduced the survival rate of A. koreana seedlings. B. velezensis CE 100 produced cell wall-degrading enzymes: chitinase, protease, and β-1,3-glucanase, and the bacterial crude enzyme fraction inhibited the spore germination and mycelial growth of P. mali and F. oxysporum in a concentration-dependent manner, causing severe morphological deformations. Consequently, treatment with B. velezensis CE 100 improved the survival rate of seedlings infected with either P. mali or F. oxysporum compared to the control and PB media treatment. The bacterium also increased nutrient availability through ammonia–nitrogen production and phosphate solubilization and produced indole-3-acetic acid (IAA), and its inoculation remarkably improved seedling growth (root and shoot dry weight) compared to the control and PB media treatment group. This is the first study to report P. mali and F. oxysporum as the pathogens causing seedling blight and wilt diseases in A. koreana nursery seedling, and the possibility of using B. velezensis CE 100 to control the fungal pathogens and improve the seedling growth without chemical use.

Multitechnique approaches for characterising phase behaviour in food biopolymer systems

SummaryUnderstanding the phase behaviour of biopolymer composites is vital for various technological applications, such as developing functional foods, nutraceuticals, encapsulation, isolation and fractionation of enzymes and proteins. The growing consumer demand for healthier food products pressures the food and nutraceutical industries to innovate while maintaining traditional texture and nutritional quality. This review critically examines various analytical techniques, including spectroscopic, microscopic, calorimetric, rheological, viscometry, and diffraction methods, used to study phase behaviour. Each technique provides unique insights into phase behaviour mechanisms at micro and macroscopic levels, with respective advantages and limitations discussed. The review advocates for a multi‐technique approach for a comprehensive understanding of phase behaviour and highlights the development of in situ probing techniques with minimal sample pre‐processing as a promising advancement. These innovations are expected to significantly enhance the practical applications of these techniques in the food industry, meeting current consumer demands without compromising traditional qualities.

Effective biocontrol efficacy of Bacillus subtilis CV21 against cherry leaf spot disease caused by Alternaria phytopathogens and growth promotion of flowering cherry (Prunus sargentii Rehder) seedlings

Cherry leaf spot (CLS) disease is one of the most common and deleterious disease of flowering cherry seedlings which lowers frost tolerance, growth, and biomass production during the season, and reduces flower production in following season. This study isolated two phytopathogens, Alternaria alternata CH3 and Alternaria alternata CH10, and confirmed their pathogenicity of CLS disease in flowering cherry seedlings, causing brownish spots and necrotic lesions on the leaves. We also isolated a plant growth-promoting bacteria (PGPB), Bacillus subtilis strain CV21, and investigated its antifungal and plant growth-promoting properties. The bacterium produced cell wall-degrading enzymes such as chitinase, β-1,3-glucanase, and protease and effectively antagonized both A. alternata CH3 and A. alternata CH10 and the crude enzyme fraction (100 µl/mL) of B. subtilis CV21 inhibited spore germination by 40.1 % and 25.1 % and reduced mycelial growth by 29.1 % and 42.5 % against A. alternata CH3 and A. alternata CH10. The crude enzyme fraction degraded the cell walls of both phytopathogens in a concentration-dependent manner, causing swelling with bulbous structures in the hyphal cell, and cell wall lysis with severe perforations, loss of shape and aggregation of spores compared to control. Treatment with the bacterial culture broth on flowering cherry seedlings reduced CLS disease by 1.3-fold and 3.4-fold compared to the chemical treatment and the control group, respectively. In addition, B. subtilis CV21 also demonstrated plant growth-promoting properties such as indole-3-acetic acid (IAA) production up to a maximum concentration of 3.5 µg/mL during the experimental period and exhibited potential for phosphate solubilization and nitrogen fixation. The inoculation of nursery seedlings with B. subtilis CV21 culture broth improved the chlorophyll content, and increased seedling growth and biomass production compared to chemical treatment and the control group. The results demonstrate that the B. subtilis CV21 could be effectively applied as a bio-fungicide to control CLS and as bio-stimulant/bio-fertilizer to enhance the growth of flowering cherry seedlings.

Effect of the Oligosaccharid Fraction of Fusarium oxysporum f. sp. vasinfectum on the Activity of Phenolic Metabolism Enzymes in Cotton Leaves

The synthesis of secondary metabolites, notably phenolic compounds, is part of the plant's defense reactions. The enzymes of phenolic metabolism can constitute a parameter of measurement of the defense of the plants. The aim of this work is to evaluate the activity of phenolic metabolism enzymes and an antioxidant enzyme after treatment of cotton plants with a fungal elicitor. Cotton plants obtained from the cultivar Y764AG3 were treated with the oligosaccharide fraction (FOS) of Fusarium oxysporum f. sp. Vasinfectum (FOV). The extraction of the enzymes is carried out cold by grinding the leaves in extraction buffer in the presence of PVP (0.5 %) and 0.1 M sodium phosphate buffer. The supernatant obtained after centrifugation of the filtrate was purified with Dowex-2 in order to obtain the purified enzymatic fraction. This fraction was used for the determination of enzyme activity by reading the absorbances. The results showed that the activity of ammonia-lyase activity was higher in the treated plants than in the control. This reflects an increase in the biosynthesis of phenolic compounds in the treated plants. On the other hand, oxidoreductase activity was higher in control plants than in treated plants. This indicates a decrease in the degradation of phenolic compounds in the treated plants. This study revealed the existence of an active phenolic metabolism in the FOS-treated plants. Thus defence mechanisms are activated in plants treated with the oligosaccharide fraction of Fusarium oxysporum f. sp. Vasinfectum.

An Enzymatic and Proteomic Analysis of Panus lecomtei during Biodegradation of Gossypol in Cottonseed.

Cotton is an important plant-based protein. Cottonseed cake, a byproduct of the biodiesel industry, offers potential in animal supplementation, although the presence of the antinutritional sesquiterpenoid gossypol limits utilization. The macrofungus Panus lecomtei offers potential in detoxification of antinutritional factors. Through an enzymatic and proteomic analysis of P. lecomtei strain BRM044603, grown on crushed whole cottonseed contrasting in the presence of free gossypol (FG), this study investigated FG biodegradation over a 15-day cultivation period. Fungal growth reduced FG to levels at 100 μg/g, with a complex adaptive response observed, involving primary metabolism and activation of oxidative enzymes for metabolism of xenobiotics. Increasing activity of secreted laccases correlated with a reduction in FG, with enzyme fractions degrading synthetic gossypol to trace levels. A total of 143 and 49 differentially abundant proteins were observed across the two contrasting growth conditions after 6 and 12 days of cultivation, respectively, revealing a dynamic protein profile during FG degradation, initially related to constitutive metabolism, then later associated with responses to oxidative stress. The findings advance our understanding of the mechanisms involved in gossypol degradation and highlight the potential of P. lecomtei BRM044603 in cotton waste biotreatment, relevant for animal supplementation, sustainable resource utilization, and bioremediation.

Changes in the structure and enzyme binding of starches during in vitro enzymatic hydrolysis using mammalian mucosal enzyme mixtures

Starches are hydrolyzed into monosaccharides by mucosal α-glucosidases in the human small intestine. However, there are few studies assessing the direct digestion of starch by these enzymes. The objective of this study was to investigate the changes in the structure and enzyme binding of starches during in vitro hydrolysis by mammalian mucosal enzymes. Waxy maize (WMS), normal maize (NMS), high-amylose maize (HAMS), waxy potato (WPS), and normal potato (NPS) starches were examined. The order of the digestion rate was different compared with other studies using a mixture of pancreatic α-amylase and amyloglucosidase. NPS was digested more than other starches. WPS was more digestible than WMS. Hydrolyzed starch from NPS, NMS, WPS, WMS, and HAMS after 24 h was 66.4, 64.2, 61.7, 58.7, and 46.2 %, respectively. Notably, a significant change in the morphology, reduced crystallinity, and a decrease in the melting enthalpy of the three starches (NPS, NMS, and WPS) after 24 h of hydrolysis were confirmed by microscopy, X-ray diffraction, and differential scanning calorimetry, respectively. The bound enzyme fraction of NPS, NMS, and WPS increased as hydrolysis progressed. In contrast, HAMS was most resistant to hydrolysis by mucosal α-glucosidases in terms of digestibility, changes in morphology, crystallinity, and thermal properties.

Frustration can Limit the Adaptation of Promiscuous Enzymes Through Gene Duplication and Specialisation

Virtually all enzymes catalyse more than one reaction, a phenomenon known as enzyme promiscuity. It is unclear whether promiscuous enzymes are more often generalists that catalyse multiple reactions at similar rates or specialists that catalyse one reaction much more efficiently than other reactions. In addition, the factors that shape whether an enzyme evolves to be a generalist or a specialist are poorly understood. To address these questions, we follow a three-pronged approach. First, we examine the distribution of promiscuity in empirical enzymes reported in the BRENDA database. We find that the promiscuity distribution of empirical enzymes is bimodal. In other words, a large fraction of promiscuous enzymes are either generalists or specialists, with few intermediates. Second, we demonstrate that enzyme biophysics is not sufficient to explain this bimodal distribution. Third, we devise a constraint-based model of promiscuous enzymes undergoing duplication and facing selection pressures favouring subfunctionalization. The model posits the existence of constraints between the catalytic efficiencies of an enzyme for different reactions and is inspired by empirical case studies. The promiscuity distribution predicted by our constraint-based model is consistent with the empirical bimodal distribution. Our results suggest that subfunctionalization is possible and beneficial only in certain enzymes. Furthermore, the model predicts that conflicting constraints and selection pressures can cause promiscuous enzymes to enter a ‘frustrated’ state, in which competing interactions limit the specialisation of enzymes. We find that frustration can be both a driver and an inhibitor of enzyme evolution by duplication and subfunctionalization. In addition, our model predicts that frustration becomes more likely as enzymes catalyse more reactions, implying that natural selection may prefer catalytically simple enzymes. In sum, our results suggest that frustration may play an important role in enzyme evolution.

Proteolytic Activity of Silkworm Thorn (Cudrania tricuspidata) Fruit for Enzymatic Hydrolysis of Food Proteins.

This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit (Cudrania tricuspidata) through ethanol precipitation at different ratios, and to determine its proteolytic activity and optimal activity conditions. Furthermore, the hydrolysis characteristics and antioxidant activity of soy protein isolate (SPI) and whey protein concentrate (WPC) hydrolyzates obtained through the enzymatic hydrolysis of freeze-dried silkworm thorn fruit powder (SF) were evaluated. For isolation and partial purification of proteolytic fraction, the water-solubilized fraction of the silkworm thorn fruit was purified through ethanol precipitation at four different ratios of 1:1, 1:2, 1:4, and 1:6 (v/v). The protein recovery rate, caseinolytic activity, protein pattern, and optimal activity (pH, temperature, and inhibitors) of fractional ethanol precipitate obtained from the silkworm thorn fruit (ESF) were evaluated. The proteolytic fraction obtained from silkworm thorn fruit exhibited a major protein band around 65-70 kDa and showed the highest proteolytic activity at a 1:4 ratio of ethanol precipitation (p < 0.05). The optimal activity of the measured enzyme fraction was determined to be at pH 9.0 and 50 °C, and the proteolytic activity of ESF was almost inhibited by phenyl methyl sulphonyl fluoride (PMSF, 2 mM), a serine protease inhibitor. Compared to Alcalase and papain, extensively used as commercial enzymes, the silkworm thorn fruit powder was less effective in hydrolyzing SPI and WPC. Nevertheless, SPI and WPC hydrolyzates mediated with silkworm thorn fruit powder showed even better antioxidant activities than those mediated with Alcalase and papain. Thus, our results show the potential application of silkworm thorn fruit as a novel source of plant protease for producing human-grade protein hydrolyzates.

Foam fractionation Tags (F-Tags) enabling surfactant-free, activity-preserving recovery of enzymes

Enzymes have become important tools in many industries. However, the full exploitation of their potential is currently limited by a lack of efficient and cost-effective methods for enzyme purification from microbial production. One technology that could solve this problem is foam fractionation. In this study, we show that diverse natural foam-stabilizing proteins fused as F-Tags to β-lactamase, penicillin G acylase, and formate dehydrogenase, respectively, are able to mediate foaming and recovery of the enzymes by foam fractionation. The catalytic activity of all three candidates is largely preserved. Under appropriate fractionation conditions, especially when a wash buffer is used, some F-Tags also allow nearly complete separation of the target enzyme from a contaminating protein. We found that a larger distance between the F-Tag and the target enzyme has a positive effect on the maintenance of catalytic activity. However, we did not identify any particular sequence motifs or physical parameters that influenced performance as an F-tag. The best results were obtained with a short helical F-Tag, which was originally intended to serve only as a linker sequence. The findings of the study suggest that the development of molecular tags that enable the establishment of surfactant-free foam fractionation for enzyme workup is a promising method.Key points• Foam-stabilizing proteins mediate activity-preserving foam fractionation of enzymes• Performance as an F-Tag is not restricted to particular structural motifs• Separation from untagged protein benefits from low foam stability and foam washingsGraphical

Antifungal and Plant-Growth Promotion Effects of Bacillus velezensis When Applied to Coastal to Pine (Pinus thunbergii Parl.) Seedlings

Fungal diseases such as root rot and leaf blight cause substantial losses in coastal pine (Pinus thunbergii Parl.) seedling production, which hinders afforestation/forest restoration programs. We isolated and identified Fusarium oxysporum and Alternaria alternata as the causal agents of root rot and needle blight diseases and investigated the biocontrol efficacy against the fungal pathogens and growth promotion of coastal pine seedlings using Bacillus velezensis CE 100. The bacterium produced the hydrolytic enzymes chitinase, β-1,3-glucanase, and protease enzymes, and the crude enzyme fraction of the biocontrol strain caused the deformation of the fungal cell wall and antagonized F. oxysporum and A. alternata, causing respective inhibition of spore germination by 91.0% and 85.9% and mycelial growth by 58.3% and 54.3%, at a concentration of 1000 µL/mL. Consequently, the bacterial treatment improved the survival rate of seedlings 1.9 times relative to the control group. The bacterium secreted indole-acetic acid (IAA) phytohormone and enhanced root growth and absorption of nutrients, which notably enhanced the biomass production of coastal pine seedlings. Therefore, these results provide evidence that B. velezensis CE 100 is an effective antifungal and growth-promoting bacterium that can facilitate the production of high-quality coastal pine seedlings for the restoration and establishment of coastal forests.

Investigation of Digestion Processes in the Stomach and Duodenum using Computational Fluid Dynamics Model

The stomach and intestines play an important role in homogenizing the contents of the tract, mixing food with enzymes. The study of digestion processes in a fed state remains difficult by experimental methods. Using computational fluid dynamics models it is possible to obtain flow characteristics at any point in the computational domain. At the previous stages of research, we developed a model to describe the flow of a multiphase multicomponent mixture in the antroduodenum, taking into account the processes of mass transfer between phases, biochemical reactions, mass sources and effluents. In this article, we propose an improvement of the model for describing the multiphase flow in the whole stomach and duodenum, taking into account the gas phase. The mathematical formulation of the problem includes the balance equations of mass and momentum for a multiphase medium with mass sources. Boundary conditions take into account the peristalsis of the tract. In numerical experiments we studied the influence of the density of food particles on the distribution of volume fractions of phases, mass fractions of food components and enzymes, pH level in the digestive tract. In the future, it is necessary to continue working with the rheological properties of the flow.

Biosoftening of banana pseudostem fiber using cellulase and pectinase enzyme isolated from Aspergillus niger for textile industry

BackgroundNowadays, farmers are facing a lot of problems for the disposal of banana pseudostem waste after the harvesting of banana. Banana pseudostem is a rich source of fiber, which is an alternative source of other natural and synthetic fibers. The banana fibers are biodegradable, and they are expected to be in great demand in the international market. For the textile industry, fibers were extracted using chemical and mechanical methods, but it leads to damage and affects the quality of fibers. So, this study mainly focused on biosoftening of banana pseudostem fiber using crude enzyme produced from Aspergillus niger which is one of the most predominant fungus which can synthesize industrially applicable enzymes and which can soften the surface of banana pseudostem fiber. Through this, biosoftened banana pseudostem fiber can be produced, and the disposal problem of banana pseudostem can be rectified in an eco-friendly manner. ResultThe present study was undertaken for the biosoftening of banana pseudostem fiber using crude enzymes isolated from fungal strain. The fungal isolates were subjected to enzyme screening such as cellulase, pectinase, chitinase, peroxidase, and polygalacturonase. The maximum production of enzyme was observed in F2 strain, and it was subjected to crude enzyme production and purification using dialysis and column chromatography. The collected best enzyme fractions were selected for the biosoftening of banana pseudostem fiber. The banana pseudostem fiber was treated with crude enzymes at the time duration of 2, 4, 24, and 48 h. After the treatment, the treated and untreated fibers were evaluated for the mechanical properties and chemical constituent’s analysis. The results revealed that the chemical contents were high during 2- and 4-h-treated fibers. After that, chemical constituents were reduced due to the removal of debris by the action of enzymes. The mechanical properties such as breaking load, breaking extension, tenacity, and diameter of fiber were best in the fibers treated for 2 and 4 h. After 4 h due to the removal of chemical constituents the breaking load and tenacity, diameter will be reduced. SEM results proved that the fiber treated at 4th h showed smooth and softened fiber. ConclusionThis study proved that the crude enzymes isolated from the Aspergillus niger can be effectively soften and increase the quality of banana pseudostem fiber.

Purification and characterization of extracellular tannase from Aspergillus fumigatus MA using Syzigium cumini leaves under solid state fermentation

This study reports the tannase purification produced by a tannery effluent-originated fungal isolate i.e., Aspergillus fumigatus MA under solid state fermentation (SSF) condition. Purification of tannase from culture filtrate was attained using ammonium sulfate precipitation with subsequent diethylaminoethyl (DEAE)-cellulose mediated ion exchange chromatographic technique. Fractional precipitation of the culture filtrate with 60–80% ammonium sulfate yielded 80.9% recovery of tannase with 6.16-fold purification. The enzyme fractions were collected and eluted as a single peak using 0.5 M NaCl-gradient concentration. DEAE-cellulose column chromatography results in overall 23-fold purification with 27.6% recovery of the enzyme. SDS-PAGE analysis of purified tannase confirmed the presence of a single band of protein with a molecular mass equivalent to 66.2 kDa. The highest activity of tannase was observed at optimum pH ranged between 5.0–6.0 whereas, the tannase stability (>80%) was observed at 4.0 to 7.0 pH ranges. The purified tannase activity was found to be optimally active at 30 °C whereas stability (>90%) was accomplished between 30–50 °C temperature. The Km and Vmax were found to be 1.61 × 10−3 M and 1.04 mM respectively. These properties suggest the potential of the enzyme to be utilized in various food, feed, and pharmaceutical sectors.

LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes

BackgroundHigh substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5–17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons’ staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions.ResultsAlthough glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55–86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25–30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining).ConclusionsThe gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

Functional proteomics analysis of Triticum durum germ

Wheat germ has been shown to contain a wide range of bioactive molecules with beneficial effects on human health. The identification of specific biomarkers could be effective in assessing the wheat germ content of wheat-based products. To date, wheat germ agglutinin (WGA), a lectin predominantly found in the germ tissue of wheat kernels, has been evaluated as a biomarker of whole-grain wheat. In this study, we performed a detailed investigation of the protein composition of wheat germ isolated from Triticum durum (Italian cultivar Svevo) with the aim of expanding the inventory of the wheat germ proteome. Wheat germ proteins were extracted and large-scale proteomics was performed using a bottom-up shotgun approach, including rough quantification using intensity Based Absolute Quantification (iBAQ). Overall, 1353 proteins were identified and functionally classified by gene ontology enrichment analysis. The predominant enzymatic fraction (280 gene products) was represented by hydrolases, including peptidases and glycosylases. Metallopeptidases were the most abundant peptidases, while the most represented glycosidases were chitinase followed by β-amylase. The identification of WGA was carried out only by a dedicated experiment based on Western blotting coupled to gel-based mass spectrometry analyses, as shotgun proteomics did not reveal this protein among those identified. This result provided a basis for a more in-depth study of the proteome in the dormant state of the germ, but also enabled to pinpoint the abundant proteins in germ that can be used as potential biomarkers as alternative to WGA.

In vitro and in vivo studies on a group of chalcones find promising results as potential drugs against fascioliasis

About a third of the world population is infected by helminth parasites implicated in foodborne trematodiasis. Fascioliasis is a worldwide disease caused by trematodes of the genus Fasciola spp. It generates huge economic losses to the agri-food industry and is currently considered an emerging zoonosis by the World Health Organization (WHO). The only available treatment relies on anthelmintic drugs, being triclabendazole (TCBZ) the drug of choice to control human infections. The emergence of TCBZ resistance in several countries and the lack of an effective vaccine to prevent infection highlights the need to develop new drugs to control this parasitosis.We have previously identified a group of benzochalcones as inhibitors of cathepsins, which have fasciolicidal activity in vitro and are potential new drugs for the control of fascioliasis. We selected the four most active compounds of this group to perform further preclinical studies. The compound's stability was determined against a liver microsomal enzyme fraction, obtaining half-lives of 34–169 min and low intrinsic clearance values (<13 μL/min/mg), as desirable for potential new drugs. None of the compounds were mutagenic or genotoxic and no in vitro cytotoxic effects were seen. Compounds C31 and C34 showed the highest selectivity index against liver fluke cathepsins when compared to human cathepsin L. They were selected for in vivo efficacy studies observing a protective effect, similar to TCBZ, in a mouse model of infection. Our findings strongly encourage us to continue the drug development pipeline for these molecules.

Method for Collection of Tear Fluid for Evaluation Its Antioxidant Properties.

A technique of collection of the tear fluid with Schirmer strips for evaluation of the activity of the main antioxidant defense enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in the tear fluid was proposed. The degree of extraction of the studied enzymes from the Schirmer strip is >85%. Cytometry showed that conjunctival and corneal cells can be transferred to the Schirmer strips during tear collection, which leads to sample contamination with intracellular fractions of the antioxidant enzymes. The approach proposed by us allows avoiding this contamination during tear fluid sampling. This technique makes it possible to increase the accuracy of determining the activity of antioxidant protection enzymes in the tear fluid and can be used for diagnostics of ocular surface pathologies in clinical practice.

Biosynthesis of silver nanoparticles using nitrate reductase from Aspergillus terreus N4 and their potential use as a non-alcoholic disinfectant.

Green technology has been developed for the quick production of stabilized silver nanoparticles (AgNPs), with the assistance of nitrate reductase from an isolated culture of Aspergillus terreus N4. The organism's intracellular and periplasmic fractions contained nitrate reductase, with the former demonstrating the highest activity of 0.20 IU/g of mycelium. When the fungus was cultivated in a medium comprising 1.056% glucose, 1.836% peptone, 0.3386% yeast extract, and 0.025% KNO3, the greatest nitrate reductase productivity of 0.3268 IU/g was achieved. Statistical modeling via response surface methodology was used to optimize the enzyme production. The periplasmic and intracellular enzyme fractions were found to convert Ag+ to Ag0, initiating synthesis within 20min, with predominant nanoparticle sizes between 25 and 30nm. By normalizing the effects of temperature, pH, AgNO3 concentration, and mycelium age with a variable shaking period for enzyme release, the production of AgNPs with the periplasmic fraction was optimized. The synthesis of nanoparticles occurred at temperatures of 30, 40, and 50°C, with the highest yield observed at 40 and 50°C during shorter incubation periods. Similarly, the nanoparticles were synthesized at pH levels of 7.0, 8.0, and 9.0, with the greatest production observed at pH 8.0 and 9.0 at lower incubation periods. The antimicrobial activity of AgNPs was demonstrated against common foodborne pathogens, including Staphylococcus aureus and Salmonella typhimurium, indicating their potential as non-alcoholic disinfectants.

Entomopathogenic Potential of Bacillus velezensis CE 100 for the Biological Control of Termite Damage in Wooden Architectural Buildings of Korean Cultural Heritage.

Biocontrol strategies are gaining tremendous attention in insect pest management, such as controlling termite damage, due to the growing awareness of the irreparable harm caused by the continuous use of synthetic pesticides. This study examines the proteolytic and chitinolytic activities of Bacillus velezensis CE 100 and its termiticidal effect through cuticle degradation. The proteolytic and chitinolytic activities of B. velezensis CE 100 systematically increased with cell growth to the respective peaks of 68.3 and 128.3 units/mL after seven days of inoculation, corresponding with the highest cell growth of 16 × 107 colony-forming units (CFU)/mL. The in vitro termiticidal assay showed that B. velezensis CE 100 caused a rapid and high rate of termite mortality, with a median lethal time (LT50) of >1 h and the highest mortality rates of 91.1% and 92.2% recorded at 11 h and 12 h in the bacterial broth culture and crude enzyme fraction, respectively. In addition to broken setae and deformed sockets, termites treated with the bacterial broth culture exhibited degraded epicuticles, while the crude enzyme fraction caused severe disintegration of both the epicuticle and endocuticle. These results indicate the tremendously higher potential of B. velezensis CE 100 in the biological control of subterranean termites compared to the previously used entomopathogenic bacteria.

Purified cellulase-mediated simultaneous sugar utilization by Bacillus albus isolated from Similipal, Odisha, India.

Among 24 isolated cellulolytic bacteria from Similipal Biosphere Reserve, the most efficient isolate was recognized as a strain of Bacillus albus. This strain of B. albus was evaluated for cellulase production and the cellulase activity was measured in submerged fermentation using substrate carboxymethyl cellulose (CMC). Different nutritional (carbon, nitrogen, and metal-ion sources) and physical variables (pH, temperature, substrate concentration, and incubation time) during the growth of B. albus were optimized to obtain maximum cellulase activity. The highest cellulase activity of 5.79 U/mL for B. albus was observed at pH 6.75, temperature 37.5°C, CMC concentration 8.5 g/L, and 42 h incubation time. Further, supplementation of glucose as a subsidiary carbon source, yeast extract, peptone as nitrogen sources, and MgSO4 and MnSO4 as metal-ion sources enhance the cellulase activity of B. albus. The purified enzyme was reported to have a molecular weight of ∼54 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A zymogram analysis evidenced the cellulase activity of the purified enzyme fractions obtained from diethylaminoethyl cellulose chromatography. The purified cellulase was reported to have an optimum pH and temperature of 7.0°C and 50°C, respectively with a capacity of retaining its 60% residual activity within pH 6.0-8.0 and temperature 30-40°C, respectively. The metal ions, K+ and Na+ were the activators, while Pb2+ and Hg2+ were the inhibitors for the purified cellulase. The purified cellulase showed Km and Vmax values of 0.38 M and 8.19 U/mL, respectively, in presence of the substrate CMC and also simultaneous consumption of both hexose and pentose sugars.