Inactivation Of Enzyme Research Articles

Generation of Plasma-Activated Fluids for Successful Disinfection of Pseudomonas aeruginosa in Liquid Environments and Determination of Microbial Damage

The application of the non-thermal atmospheric pressure plasma technology is a promising tool for microbial inactivation. During the activation process, many reactive substances and radicals arise associated with physicochemical changes in the fluid and massive pH drop. In this study, we analyzed and optimized plasma activation settings and conditions of water and liquids to obtain inactivation of the waterborne microorganism Pseudomonas aeruginosa in a liquid environment. The minimal electrical output was 60 Watt with 20 min activation time followed by 30 min contact time with 108 cells/mL. Using higher electrical power (>90 W) with a Lab Unit generating plasma-activated water, a shorter activation time (<10 min) was sufficient for bacterial inactivation. The organic and inorganic composition of the activated liquid with different mineral salt concentrations is of utmost importance for the yield of reactive species during the plasma activation process and consequently for the antimicrobial effect. Plasma-activated fluids with high organic and inorganic contents demonstrated lower inactivation efficiencies than low loaded fluids; yet antimicrobial efficacy could be achieved by increasing the electrical power and activation time. For sufficient inactivation of bacterial suspensions, at least half a volume unit of plasma-activated water had to be added after appropriately optimized activation. Further dilutions reduced the antimicrobial effect. PAW lost activity after being left standing for a prolonged time after activation, so for maximizing the antimicrobial effect a direct use after activation is recommendable. Bacterial inactivation was shown by the absence of colony forming units on culture media and, at the molecular level, damage to the membrane and inactivation of enzymes were observed. Plasma-activated fluids demonstrated a high potential in applications as microbiological disinfectant in liquids.

MitoCur-1 induces ferroptosis to reverse vemurafenib resistance in melanoma through inhibition of USP14.

Melanoma is an aggressive malignant tumor with a poor prognosis. Vemurafenib (PLX4032, vem) is applied to specifically treat BRAF V600E-mutated melanoma patients. However, prolonged usage of vem makes patients resistant to the drug and finally leads to clinical failure. We previously tested the combination regimen of tubulin inhibitor VERU-111 with vem, as well as USP14 selective inhibitor b-AP15 in combination with vem, both of which have showed profound therapeutic effects in overcoming vem resistance invitro and invivo. Most importantly, we discovered that vem-resistant melanoma cell lines highly expressed E3 ligase SKP2 and DUB enzyme USP14, and we have demonstrated that USP14 directly interacts and stabilizes SKP2, which contributes to vem resistance. These works give us a clue that USP14 might be a promising target to overcome vem resistance in melanoma. MitoCur-1 is a curcumin derivative, which was originally designed to specifically target tumor mitochondria inducing redox imbalance, thereby promoting tumor cell death. In this study, we have demonstrated that it can work as a novel USP14 inhibitor, and thus bears great potential in providing an anti-tumor effect and sensitizing vem-resistant cells by inducing ferroptosis in melanoma. Application of MitoCur-1 dramatically induces USP14 inhibition and inactivation of GPX4 enzyme, meanwhile, increases the depletion of GSH and decreases SLC7A11 expression level. As a result, ferrous iron-dependent lipid ROS accumulated in the cell, inducing ferroptosis, thus sensitizes the vem-resistant melanoma cell. Interestingly, overexpression of USP14 antagonized all the ferroptosis cascade events induced by MitoCur-1, therefore, we conclude that MitoCur-1 induces ferroptosis through inhibition of USP14. We believe that by inhibition of USP14, vem resistance can be reversed and will finally benefit melanoma patients in future.

Chitosan-based intelligent polymeric networks for site-specific colon medication delivery: A comprehensive study on controlled release of diloxanide furoate and network formation dynamics

Oral medications are prone to gastric degradation and enzymatic inactivation, diminishing their efficacy. This study investigates a solution by developing intelligent polymeric networks, incorporating chitosan, methacrylic acid, N, N, methylene bisacrylamide, and montmorillonite clay, to enable the controlled release of Diloxanide Furoate (DF), an anti-protozoal drug. Employing a swelling-assisted diffusion technique, drug loading percentages varied from 63.96 % to 76.82 % among different formulations. Increased chitosan and methacrylic acid content enhanced drug loading, while N, N, methylene bisacrylamide and montmorillonite clay demonstrated an inverse relationship affecting diffusion and swelling.Equilibrium swelling studies unveiled formulation-dependent behaviors, with chitosan reducing swelling and methacrylic acid promoting it. Higher N, N, methylene bisacrylamide concentrations decreased swelling, indicating a denser cross-linked structure, while montmorillonite clay reduced hydrophilicity and swelling capacity. Further analyses confirmed successful gel formation, particularly in formulations with higher chitosan, methacrylic acid, and N, N, methylene bisacrylamide content, while montmorillonite clay limited gel fraction due to restricted polymer chain mobility. Techniques such as Fourier transform infrared spectroscopy, Differential scanning calorimetry, and thermal gravimetric analyses supported network development, enhancing thermal stability and cross-linking density. This research underscores the flexibility of polymeric networks for precise drug delivery, offering potential advancements in targeted therapies for various medical conditions.

Bioactive ZnO Nanoparticles: Biosynthesis, Characterization and Potential Antimicrobial Applications.

In recent years, biosynthesized zinc oxide nanoparticles (ZnONPs) have gained tremendous attention because of their safe and non-toxic nature and distinctive biomedical applications. A diverse range of microbes (bacteria, fungi and yeast) and various parts (leaf, root, fruit, flower, peel, stem, etc.) of plants have been exploited for the facile, rapid, cost-effective and non-toxic synthesis of ZnONPs. Plant extracts, microbial biomass or culture supernatant contain various biomolecules including enzymes, amino acids, proteins, vitamins, alkaloids, flavonoids, etc., which serve as reducing, capping and stabilizing agents during the biosynthesis of ZnONPs. The biosynthesized ZnONPs are generally characterized using UV-VIS spectroscopy, TEM, SEM, EDX, XRD, FTIR, etc. Antibiotic resistance is a serious problem for global public health. Due to mutation, shifting environmental circumstances and excessive drug use, the number of multidrug-resistant pathogenic microbes is continuously rising. To solve this issue, novel, safe and effective antimicrobial agents are needed urgently. Biosynthesized ZnONPs could be novel and effective antimicrobial agents because of their safe and non-toxic nature and powerful antimicrobial characteristics. It is proven that biosynthesized ZnONPs have strong antimicrobial activity against various pathogenic microorganisms including multidrug-resistant bacteria. The possible antimicrobial mechanisms of ZnONPs are the generation of reactive oxygen species, physical interactions, disruption of the cell walls and cell membranes, damage to DNA, enzyme inactivation, protein denaturation, ribosomal destabilization and mitochondrial dysfunction. In this review, the biosynthesis of ZnONPs using microbes and plants and their characterization have been reviewed comprehensively. Also, the antimicrobial applications and mechanisms of biosynthesized ZnONPs against various pathogenic microorganisms have been highlighted.

Electrostatic interactions of enzymes in non-aqueous conditions: insights from molecular dynamics simulations

Electrostatic interactions of enzymes and their effects on enzyme activity and stability are poorly understood in non-aqueous conditions. Here, we investigate the contribution of the electrostatic interactions on the stability and activity of enzymes in the non-aqueous environment using molecular dynamics simulations. Lipase was selected as active and lysozyme as inactive model enzymes in non-aqueous media. Hexane was used as a common non-aqueous solvent model. In agreement with the previous experiments, simulations show that lysozyme has more structural instabilities than lipase in hexane. The number of hydrogen bonds and salt bridges of both enzymes is dramatically increased in hexane. In contrast to the other opinions, we show that the increase of the electrostatic interactions in non-aqueous media is not so favorable for enzymatic function and stability. In this condition, the newly formed hydrogen bonds and salt bridges can partially denature the local structure of the enzymes. For lysozyme, the changes in electrostatic interactions occur in all domains including the active site cleft, which leads to enzyme inactivation and destabilization. Interestingly, most of the changes in electrostatic interactions of lipase occur far from the active site regions. Therefore, the active site entrance regions remain functional in hexane. The results of this study reveal how the changes in electrostatic interactions can affect enzyme stability and activity in non-aqueous conditions. Moreover, we show for the first time how some enzymes, such as lipase, remain active in a non-aqueous environment. Communicated by Ramaswamy H. Sarma

EVALUATION OF SOYBEAN FLOURS OBTAINED FROM WHOLE AND SHELLED GRAINS SUBJECTED TO HEAT TREATMENT

This study aimed to obtain and characterize soy flours from whole (WG) and shelled grains (SG) of the conventional cultivar Vmax and special cultivars used for human consumption, BRS 267 and BRS 257. For lipoxygenase enzyme inactivation, the WGs and SGs of BRS 267 and Vmax were bleached at 98 °C for 5 min, roasted at 150 °C for 30 min, milled and finally sieved through a size 40 mesh to obtain the flours. These flours were analyzed for proteins, lipids, isoflavones, Kunitz trypsin inhibitor, their nitrogen solubility index and their protein dispersibility index. The grain heat treatments caused a reduction in protein solubility, an increase in aglycone isoflavones and an 80% reduction in levels of the Kunitz trypsin inhibitor. Flours from bleached WGs were brown in color. Flours from BRS 267 had higher protein levels, while those from the cultivar Vmax had the highest lipid content. Flours from BRS 257 had greater protein solubility. Thus, varieties of special soybeans, genetically enhanced for human consumption, can be processed for better quality soy foods, promoting their acceptance.

Development of thermoultrasound assisted blanching to improve enzyme inactivation efficiency, drying characteristics, energy consumption, and physiochemical properties of sweet potatoes

Thermoultrasound (USB) as a promising alternative to traditional hot water (HWB) blanching was employed to blanch sweet potatoes and its influence on enzyme activity, drying behavior, energy consumption and physiochemical properties of sweet potatoes were investigated. Results showed that successive increases in blanching temperature and time resulted in significant (p < 0.05) decreases in PPO and POD activities. Compared to HWB, USB led to more effective drying by promoting texture softening, moisture diffusion, microstructure alterations, and microchannels formation, which significantly reduced energy consumption and improved the overall quality of the dried sample. Specifically, USB at 65 °C for 15 min improved water holding capacity and ABTS, while USB at 65 °C for 30 min improved color (more red and yellow), total phenolic content, total carotenoid content, and DPPH. Unfortunately, blanching process showed detrimental effects on the amino acid composition of dried samples. Overall, the development of thermoultrasound assisted blanching for sweet potatoes has the potential to revolutionize the processing and production of high-quality sweet potato products, while also improving the sustainability of food processing operations.

New pulsed electric fields approach to improve the blanching of carrots

Pulsed Electric Fields (PEF)-ohmic is one of the most recently proposed alternatives for the application of ohmic heating in order to provide a more uniform and faster heating of food. In this study, the application of PEF-ohmic (1.33 kV/cm, 100 Hz) for the blanching of carrot cylinders was explored, and the inactivation of the peroxidase enzyme was mathematically simulated and experimentally validated. The effect of this PEF-ohmic treatment on carrot texture and its effect on the in vitro bioaccessibility of β-carotene were also determined. The best heating uniformity was achieved when PEF-ohmic was applied to carrots immersed in water at a starting temperature of 80 °C and after applying brief heating-up treatments (90 s). However, a holding heating phase (85 °C for 50 s) was required to achieve complete peroxidase inactivation after the heating-up PEF phase. The blanching time was reduced by 60 % without negatively affecting the texture of the carrots and slightly increasing (3.9 %) the bioaccessibility of the β-carotene. Overall, PEF could be an effective system for rapid and volumetric blanching of vegetables.

Response surface optimization of pulsed electric field processed kiwi–carrot juice: enzyme inactivation and evaluation of physicochemical and nutritional properties

Response surface optimization of pulsed electric field processed kiwi–carrot juice: enzyme inactivation and evaluation of physicochemical and nutritional properties

Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Kinetic and structural details of urease inactivation by thiuram disulphides

This paper reports on the molecular details of the reactivity of urease, a nickel-dependent enzyme that catalyses the last step of organic nitrogen mineralization, with thiuram disulphides, a class of molecules known to inactivate the enzyme with high efficacy but for which the mechanism of action had not been yet established. IC50 values of tetramethylthiuram disulphide (TMTD or Thiram) and tetraethylthiuram disulphide (TETD or Disulfiram) in the low micromolar range were determined for plant and bacterial ureases. The X-ray crystal structure of Sporosarcina pasteurii urease inactivated by Thiram, determined at 1.68 Å resolution, revealed the presence of a covalent modification of the catalytically essential cysteine residue. This is located on the flexible flap that modulates the size of the active site channel and cavity. Formation of a Cys-S-S-C(S)-N(CH3)2 functionality responsible for enzyme inactivation was observed. Quantum-mechanical calculations carried out to rationalise the large reactivity of the active site cysteine support the view that a conserved histidine residue, adjacent to the cysteine in the active site flap, modulates the charge and electron density along the thiol SH bond by shifting electrons towards the sulphur atom and rendering the thiol proton more reactive. We speculate that this proton could be transferred to the nickel-coordinated urea amide group to yield a molecule of ammonia from the generated Curea-NH3+ functionality during catalysis.

Emerging biotechnologies and non-thermal technologies for winemaking in a context of global warming.

In the current situation, wine areas are affected by several problems in a context of global warming: asymmetric maturities, pH increasing, high alcohol degree and flat wines with low freshness and poor aroma profile. The use of emerging biotechnologies allows to control or manage such problems. Emerging non-Saccharomyces as Lachancea thermotolerans are very useful for controlling pH by the formation of stable lactic acid from sugars with a slight concomitant alcohol reduction. Lower pH improves freshness increasing simultaneously microbiological stability. The use of Hanseniaspora spp. (specially H. vineae and H. opuntiae) or Metschnikowia pulcherrima promotes a better aroma complexity and improves wine sensory profile by the expression of a more complex metabolic pattern and the release of extracellular enzymes. Some of them are also compatible or synergic with the acidification by L. thermotolerans, and M. pulcherrima is an interesting biotool for reductive winemaking and bioprotection. The use of bioprotection is a powerful tool in this context, allowing oxidation control by oxygen depletion, the inhibition of some wild microorganisms, improving the implantation of some starters and limiting SO2. This can be complemented with the use of reductive yeast derivatives with high contents of reducing peptides and relevant compounds such as glutathione that also are interesting to reduce SO2. Finally, the use of emerging non-thermal technologies as Ultra High-Pressure Homogenization (UHPH) and Pulsed Light (PL) increases wine stability by microbial control and inactivation of oxidative enzymes, improving the implantation of emerging non-Saccharomyces and lowering SO2 additions. GRAPHICAL ABSTRACT.

Effect of High-Powered Ultrasound on Bioactive Compounds and Microbiological Stability of Juices—Review

Extending the shelf life of fruit and vegetable juices plays a key role in improving food security. Ultrasonic technology can be an effective method in the process of replacing traditional heat treatment. It offers a number of benefits, such as better product quality expressed as a higher content of bioactive substances and the preservation of the natural sensory characteristics of the juice: consistency, colour, taste and smell. This paper discusses the methods of ultrasound generation, the equipment used and advances in the construction of ultrasound devices. The influence of ultrasounds on the physical and chemical properties of juices was also analysed, with particular emphasis on bioactive substances and the effectiveness of ultrasonic treatment in the inactivation of microorganisms and enzymes. Prospects and trends in the development of ultrasonic techniques that can effectively increase the efficiency of the ultrasonic juice preservation process are also described. Sonication used alone or with other processing techniques makes it possible to achieve a positive effect on the quality of consumed fruit and vegetable juices.

Role of sugars in the inactivation of horseradish peroxidase induced by cold atmospheric plasma

This study investigated the effect of different CAP exposures on the activity of horseradish peroxidase (HRP) in phosphate buffer and in model systems with different concentrations of monosaccharides (glucose, fructose) and disaccharides (sucrose, trehalose) to evaluate the potential role of sugars in enzyme inactivation. Spectroscopic analyses (fluorescence, circular dichroism, UV–Vis absorption) were conducted to highlight structural modifications possibly responsible for HRP inactivation. In phosphate buffer, the inactivation of HRP by CAP resulted dependent on both the treatment time (up to 70% after 30 min) and ozone concentration in the chamber. HRP residual activity was fitted with a very good approximation by the Weibull model. Sugars reduced the CAP efficacy, and this effect was concentration-dependent and much higher for disaccharides than monosaccharides. UV and fluorescence spectra showed that sugars differently preserved HRP tertiary structure, hindering heme degradation and quenching of aromatic amino acids. Likewise, sugars differently affected the loss of secondary structures. These findings only partially explained the diverse protective effects of sugars on HRP. Other factors, such as the different sugars’ abilities to quench plasma reactive species, reduce the system mobility, and stabilize proteins by preserving their hydration shell, were retained to play a role in the reduction of HRP inactivation. This study deepened the knowledge of the effect of CAP on peroxidase activity and demonstrated that the presence of mono and disaccharides, naturally found in fruits and vegetables and widely employed in food and beverage industries, can mitigate the inactivation and structural alterations induced by CAP on peroxidase enzymes.

Mechanisms of antimicrobial resistance of Staphylococcus aureus strains: narrative synthesis

One of the causes of the increasing incidence in recent years, worldwide, of bacterial infections resulting in therapeutic failure is the unjustified use of antimicrobials and the dissemination of antibiotic resistance factors. Currently, infections, caused by Staphylococcus aureus appear to be caused by a pathogen multiresistant to a very wide range of antimicrobials used in medical practice. A narrative synthesis study was conducted. The analysis of publications between 2012-2022 was carried out regarding the theoretical aspects of the antimicrobial resistance mechanisms characteristic of Staphylococcus aureus strains, by using the terms "Resistance mechanisms", "Resistance genes", "Staphylococcus aureus", "Antimicrobial preparations". The initial search yielded 44 articles, from which 27 eligible papers were selected and analyzed. S. aureus is one of the most important pathogens in terms of antimicrobial resistance, as it has been able to develop resistance mechanisms to almost all preparations used against it. This species can easily exemplify adaptive evolution to antimicrobials, as it has demonstrated a unique ability to rapidly respond to each new antibiotic by developing a resistance mechanism, starting with penicillin and methicillin, and ending with the newer ones, linezolid and daptomycin. The main mechanisms of S. aureus resistance to antimicrobials are enzymatic inactivation of the antibiotic, modification of the attack target, antibiotic uptake and efflux pumps. Antimicrobial resistance mechanisms are very diverse and can be intrinsic or acquired, therefore understanding them can create new treatment alternatives for infectious pathology and facilitate the development of new antimicrobials that will counteract the attempts of microorganisms to become resistant.

B-334 How to Prevent the Next Wave in Overdose Crisis?—Detect Designer Benzodiazepines Using CEDIA and DRI Assays

Abstract Background Benzodiazepines are a class of drugs used for relieving symptoms of anxiety, insomnia, agitation, muscle spasms, and alcohol withdrawal. Benzodiazepines are CNS depressants enhancing the effect of GABA neurotransmitter at GABA receptors and they are well-known for drug abuse potential and drug dependence.Designer benzodiazepines are new psychoactive substances emerging in the past two decades. Designer benzodiazepines are often taken by individuals who use hallucinogenic and stimulant drugs. The drug abuse can lead to respiratory depression, coma, or death. There exists a need to develop a new homogeneous enzyme immunoassay. Existing commercial immunoassay antibodies may also cross-react with designer benzodiazepines which have chemical structure closely related to classic therapeutic benzodiazepines. Herein, we evaluated designer benzodiazepines cross-reactivity using DRI Benzodiazepines and CEDIA Benzodiazepines assays and demonstrated the potential of detecting designer benzodiazepines in urines. Methods DRI assay is based on competition between drug labeled with glucose-6-phosphate dehydrogenase (G6PDH) and free drug in the urine sample for a fixed amount of antibody binding sites. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert NAD to NADH. CEDIA technology is based on the bacterial enzyme β-galactosidase which has been genetically engineered into two inactive fragments, Enzyme Acceptor (EA) and Enzyme Donor (ED). The enzyme activity is then determined spectrophotometrically at 570 nm. Designer Benzodiazepines tested in this study are 3-Hydroxyphenazepam, Adinazolam, Bromazolam, Clonazolam, Cloniprazepam, Deschloroetizolam, Diclazepam, Etizolam, Flubromazepam, Flubromazolam, Flunitrazolam, Meclonazepam, N-Desmethylflunitrazepam, Nifoxipam, Nimetazepam, Nitrazolam, and Pyrazolam. Compounds were spiked into drug free urine and cross-reactivity was evaluated using DRI® Benzodiazepine Assay and CEDIA® Benzodiazepine Assays on the Beckman Coulter AU680. Results 14 out of 17 (82%) tested designer benzodiazepines showed high cross-reactivity (&amp;gt;100%) in DRI benzodiazepine assay. 12 out of 17 (71%) tested designer benzodiazepines showed high cross-reactivity (&amp;gt;100%) in CEDIA assays with 200 ng/mL cutoff and 300 ng/mL cutoff. Compared to NPS-benzodiazepines trend data, this study demonstrated that DRI and CEDIA benzodiazepine assays can detect more than 50% (10 out of 19) designer benzodiazepines over 2020 Q1 to 2022 Q4 in the United States. Conclusion DRI and CEDIA Benzodiazepine Assays showed high cross reactivity of most seventeen designer benzodiazepines. The results provide potential tools for detect emerging designer benzodiazepines. Note: This was preliminary data that has not been reviewed by any regulatory authorities/cleared by FDA.

Enhancement of physicochemical stability and reduction in enzyme and microbial activity of apple juice by hydrodynamic cavitation processing

Conventional thermally processed apple juices are nutritionally depleted because the heat sensitivity degrades the bioactive compounds that comprise them. This study proposed a non-thermal technique, hydrodynamic cavitation (HC), to preserve the nutritional content and prevent the loss of bioactive components in apple juice. In addition, the effect of hydrodynamic cavitation on the physicochemical, nutrition, and enzyme inactivation of freshly expressed apple juice has been studied. The design expert software has been used for process optimization, while fuzzy logic was applied for sensory. Hydrodynamic cavitation has been used on freshly extracted apple juice with inlet pressure in the range of (34.47–103.421 kPa) for a treatment time of (0–30 min). The apple juice was treated thermally at 90 °C for 2.5 min to compare the cavitation results. The settling of particles in the cavitation sample was 7%, which was lower than the heat-treated sample (10%) and untreated control sample (39%) and had a noticeable effect on the cloud value and stability of the apple juice. The HC treatment reduces the particle size of fresh apple juice from 8934.09 nm to 1112 nm, resulting in a homogenizing effect, a decrease in viscosity, and improved juice stability. Hydrodynamic cavitation reduced the antioxidant activity from 0.4987 mg GAE mL−1 (control) to 0.3951 mg GAE mL−1 yet retained better activity than the heat-treated apple juice (0.3489 ± 0.34 mg GAE mL−1) with microbial log reduction of 0.9 at 103.42 kPa treated for 30 min. After 15 days of storage at 4 °C, HC-treated juice had higher quality features and nutrient retention than heat-treated apple juice.

A Malassezia pseudoprotease dominates the secreted hydrolase landscape and is a potential allergen on skin

Malassezia globosa is abundant and prevalent on sebaceous areas of the human skin. Genome annotation reveals that M. globosa possesses a repertoire of secreted hydrolytic enzymes relevant for lipid and protein metabolism. However, the functional significance of these enzymes is uncertain and presence of these genes in the genome does not always translate to expression at the cutaneous surface. In this study we utilized targeted RNA sequencing from samples isolated directly from the skin to quantify gene expression of M. globosa secreted proteases, lipases, phospholipases and sphingomyelinases. Our findings indicate that the expression of these enzymes is dynamically regulated by the environment in which the fungus resides, as different growth phases of the planktonic culture of M. globosa show distinct expression levels. Furthermore, we observed significant differences in the expression of these enzymes in culture compared to healthy sebaceous skin sites. By examining the in situ gene expression of M. globosa's secreted hydrolases, we identified a predicted aspartyl protease, MGL_3331, which is highly expressed on both healthy and disease-affected dermatological sites. However, molecular modeling and biochemical studies revealed that this protein has a non-canonical active site motif and lacks measurable proteolytic activity. This pseudoprotease MGL_3331 elicits a heightened IgE-reactivity in blood plasma isolated from patients with atopic dermatitis compared to healthy individuals and invokes a pro-inflammatory response in peripheral blood mononuclear cells. Overall, our study highlights the importance of studying fungal proteins expressed in physiologically relevant environments and underscores the notion that secreted inactive enzymes may have important functions in influencing host immunity.

Inactivation of the Sts enzymes promotes resistance to lethal Staphylococcus aureus infection.

Staphylococcus aureus is a highly infective Gram-positive bacterial pathogen that causes a wide range of diseases in both healthy and immunocompromised individuals. It can evade host immune defenses by expressing numerous virulence factors and toxins. Coupled with the inability of the human host to develop protective immunity against S. aureus, the emergence of antibiotic-resistant strains complicates treatment options. The non-canonical Sts phosphatases negatively regulate signaling pathways in varied immune cell types. To determine the role of the Sts proteins in regulating host responses to a Gram-positive microorganism, we investigated the response of mice lacking Sts expression to S. aureus infection. Herein, we demonstrate that Sts -/- animals are significantly resistant to lethal intravenous doses of S. aureus strain USA300. Resistance is characterized by significantly enhanced survival and accelerated bacterial clearance in multiple peripheral organs. Infected Sts -/- animals do not display increased levels of cytokines TNFα, IFNγ, and IL-6 in the spleen, liver, and kidney during the early stages of the infection, suggesting that a heightened pro-inflammatory response does not underlie the resistance phenotype. In vivo ablation of mononuclear phagocytes compromises the Sts -/- enhanced CFU clearance phenotype. Additionally, Sts -/- bone marrow-derived macrophages demonstrate significantly enhanced restriction of intracellular S. aureus following ex vivo infection. These results reveal the Sts enzymes to be critical regulators of host immunity to a virulent Gram-positive pathogen and identify them as therapeutic targets for optimizing host anti-microbial responses.

Conserved residues at the family and subfamily levels determine enzyme activity and substrate binding in glycoside hydrolase family 13

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering.