Hydrolytic Activity Research Articles

Characterization of probiotics isolated from dietary supplements and evaluation of metabiotic-antibiotic combinations as promising therapeutic options against antibiotic-resistant pathogens using time-kill assay

BackgroundThe global probiotics dietary supplements market size is continuously growing. To overcome probiotics’ health concerns, metabiotics are recognized as a safer alternative. Aiming to deal with the escalating antimicrobial resistance, the current work demonstrates synergistic metabiotic-antibiotic combinations against antibiotic-resistant pathogens.MethodsThe probiotic properties of lactic acid bacteria (LAB) strains isolated from 3 commercial dietary supplements were characterized in vitro. The combinations of the cell-free supernatants (CFS) of selected probiotic strains and conventional antibiotics against Staphylococcus aureus and Escherichia coli clinical isolates were evaluated using the time-kill assay. To our knowledge, the current literature lacks sufficient time-kill assay studies revealing the kinetics of such metabiotic-antibiotic combinations against S. aureus and E. coli.ResultsFour LAB strains isolated from dietary supplements as well as two reference strains were included in this study. The isolated LAB strains were identified by MALDI-TOF mass spectrometry as follows: P2: Lactobacillus acidophilus, P3: Lactiplantibacillus plantarum, P4: Lacticaseibacillus rhamnosus, and P5: Pediococcus acidilactici. The identification matched with that annotated by the manufacturers, except for P3. The tested strains could resist the acidic environment at pH 3. Excluding P2, the examined strains showed less than 1 log reduction in survivors upon the addition of reconstituted skimmed milk to pepsin at pH 2 and displayed an acceptable tolerance to 0.3% ox-bile. All the strains tolerated pancreatin. The hydrophobicity and autoaggregation capacities ranged between 7–92% and 36–66%, respectively. P2 was excluded owing to its inferior probiotic potential. Although the remaining strains showed excellent growth at 0.2% phenol, their growth was reduced at higher concentrations. L. plantarum and P. acidilactici strains possessed bile salt hydrolysis activity. The time-kill assay revealed promising synergistic activities of the combinations of CFS of L. rhamnosus P4 with either ceftazidime or gentamicin against E. coli and with only ceftazidime against S. aureus, as well as CFS of P. acidilactici P5 and ceftazidime against S. aureus.ConclusionsStrict identification and evaluation of the probiotic strains incorporated in dietary supplements is crucial to ensure their safety and efficacy. The CFS of probiotics could be utilized to formulate novel biotherapeutics targeting problematic pathogens. However, future in vivo studies are required to evaluate the appropriate treatment regimen.

Investigation of Antioxidant Activity of Protein Hydrolysates from New Zealand Commercial Low-Grade Fish Roes.

The objective of this study was to investigate the nutrient composition of low-grade New Zealand commercial fish (Gemfish and Hoki) roe and to investigate the effects of delipidation and freeze-drying processes on roe hydrolysis and antioxidant activities of their protein hydrolysates. Enzymatic hydrolysis of the Hoki and Gemfish roe homogenates was carried out using three commercial proteases: Alcalase, bacterial protease HT, and fungal protease FP-II. The protein and lipid contents of Gemfish and Hoki roes were 23.8% and 7.6%; and 17.9% and 10.1%, respectively. The lipid fraction consisted mainly of monounsaturated fatty acid (MUFA) in both Gemfish roe (41.5%) and Hoki roe (40.2%), and docosahexaenoic (DHA) was the dominant polyunsaturated fatty acid (PUFA) in Gemfish roe (21.4%) and Hoki roe (18.6%). Phosphatidylcholine was the main phospholipid in Gemfish roe (34.6%) and Hoki roe (28.7%). Alcalase achieved the most extensive hydrolysis, and its hydrolysate displayed the highest 2,2-dipheny1-1-picrylhydrazyl (DPPH)˙ and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidant power (FRAP). The combination of defatting and freeze-drying treatments reduced DPPH˙ scavenging activity (by 38%), ABTS˙ scavenging activity (by 40%) and ferric (Fe3+) reducing power by18% (p < 0.05). These findings indicate that pre-processing treatments of delipidation and freeze-drying could negatively impact the effectiveness of enzymatic hydrolysis in extracting valuable compounds from low grade roe.

Exploring structural features of sleep-enhancing peptides derived from casein hydrolysates by chemometrics and random forest methodology

Milk casein is regarded as source to release potential sleep-enhancing peptides. Although various casein hydrolysates exhibited sleep-enhancing activity, the underlying reason remains unclear. This study firstly revealed the structural features of potential sleep-enhancing peptides from casein hydrolysates analyzed through peptidomics and multivariate analysis. Additionally, a random forest model and a potential Tyr-based peptide library were established, and then those peptides were quantified to facilitate rapidly-screening. Our findings indicated that YP-, YI/L, and YQ-type peptides with 4–10 amino acids contributed more to higher sleep-enhancing activity of casein hydrolysates, due to their crucial structural features and abundant numbers. Furthermore, three novel strong sleep-enhancing peptides, YQKFPQY, YPFPGPIPN, and YIPIQY were screened, and their activities were validated in vivo. Molecular docking results elucidated the importance of the YP/I/L/Q- structure at the N-terminus of casein peptides in forming crucial hydrogen bond and π-alkyl interactions with His-102 and Asn-60, respectively in the GABAA receptor for activation.

Combatting Antibiotic-Resistant Pathogens: Disrupting Pseudomonas Aeruginosa Biofilm Viscoelasticity Using Carbon Dot Nanoparticles

Bacterial infection is a global burden, killing one in every eight people worldwide, primarily due to antibiotic resistance. Pseudomonas aeruginosa is one of the top five bacteria responsible for around 7.7 million fatalities caused by bacterial infections. P. aeruginosa secretes biofilm, a biological substance with viscoelastic characteristics to avoid antimicrobial treatments particularly, in chronic and serious wound infections. In the bid to create topical treatments, we investigated the mechano-active potentials of cellulase-based carbon dots as a therapeutic agent against P. aeruginosa biofilm where we envisage the retaining of the cellulase hydrolytic activity in the cellulase-based carbon dots. Cellulase is a glycoside hydrolase that has already been shown to be effective in causing biofilm dispersal as well as reduction in biofilm stiffness and elasticity.The cellulase carbon dots were synthesized using the microwave while citric acid carbon dots (CA-CDs) were hydrothermally synthesized to serve as non-protein-based carbon dots and then characterized for surface properties, size, elemental compositions, and fluorescence profile. P. aeruginosa biofilms were grown in a microfluidic environment for 24 hours and then treated with these carbon dots at a range of concentrations. Using microrheology, it was seen that biofilms treated with cellulase-based carbon dots were more viscous and less stiff compared to those treated with citric acid-based carbon dots. This appears to be because the cellulase carbon dots impact PSL within the biofilm based on microrheology studies examining bacteria strains with specific EPS components removed. This is consistent with the impact of molecular cellulase on biofilms. In general, initial studies suggest that the cellulase-based carbon dots have the potential to act as an excellent multi-antibiofilm therapeutic in the future.

(Invited) Single-Walled Carbon Nanotube Functionalization Strategies for Monitoring Enzymatic Activity and Inhibition

Semiconducting single-walled carbon nanotubes (SWCNTs) fluoresce in the near-infrared range, which overlaps with the transparency window of biological samples, and they do not photobleach or blink. Moreover, they benefit from biocompatibility and the large surface area available for functionalization. Using tailored surface functionalization, SWCNTs can be rendered optical nanosensors, such that surface binding events or changes in the local proximity of the nanotubes translate to a modulation of the emitted fluorescence. This approach was successfully used to demonstrate the detection of small molecules, volatiles, bacteria, microRNA, proteins, metals, self-assembly processes, and active processes in vivo. We will discuss different strategies for monitoring enzymatic activity and inhibition using SWCNT-based sensors, including the incorporation of the enzyme’s target bond within a synthetic molecular complex used for suspending the SWCNTs, or by the detection of the product of the enzyme hydrolytic activity. We will also highlight recent applications of these techniques in clinically relevant samples. These findings not only showcase the potential of near-infrared fluorescent SWCNT in tracking enzymatic activity but also underscore their promising role in advancing biomedical research and applications.

Enhancement of the degradation capacity of IsPETase by acidic amino acids insertion and carbohydrate-binding module fusion.

The biocatalytic degradation of poly(ethylene terephthalate) (PET) through enzymatic methods has garnered considerable attention due to its environmentally friendly and non-polluting nature, as well as its high specificity. While previous efforts in enhancing IsPETase performance have focused on amino acid substitutions in protein engineering, we introduced an amino acid insertion strategy in this work. By inserting a negatively charged acidic amino acid, Glu, at the right-angle bend of IsPETase, the binding capability between the enzyme's active pocket and PET was improved. The resulted mutant IsPETase9394insE exhibited enhanced hydrolytic activity towards PET at various temperatures ranging from 30 to 45 ℃ compared with the wild-type IsPETase. Notably, a 10.04-fold increase was observed at 45 ℃. To further enhance PET hydrolysis, different carbohydrate-binding modules (CBMs) were incorporated at the C-terminus of IsPETase9394insE. Among these, the fusion of CBM from Verrucosispora sioxanthis exhibited the highest enhancement, resulting in a 1.82-fold increase in PET hydrolytic activity at 37 ℃ compared with the IsPETase9394insE. Finally, the engineered variant was successfully employed for the degradation of polyester filter cloth, demonstrating its promising hydrolytic capacity. In conclusion, this research presents an alternative enzyme engineering strategy for modifying PETases and enriches the pool of potential candidates for industrial PET degradation.

Aspergillus niger Ochratoxinase Is a Highly Specific, Metal-Dependent Amidohydrolase Suitable for OTA Biodetoxification in Food and Feed.

Microbial enzymes can be used as processing aids or additives in food and feed industries. Enzymatic detoxification of ochratoxin A (OTA) is a promising method to reduce OTA content. Here, we characterize the full-length enzyme ochratoxinase (AnOTA), an amidohydrolase from Aspergillus niger. AnOTA hydrolyzes OTA and ochratoxin B (OTB) mycotoxins efficiently and also other substrates containing phenylalanine, alanine, or leucine residues at their C-terminal position, revealing a narrow specificity profile. AnOTA lacks endopeptidase or aminoacylase activities. The structural basis of the molecular recognition by AnOTA of OTA, OTB, and a wide array of model substrates has been investigated by molecular docking simulation. AnOTA shows maximal hydrolytic activity at neutral pH and high temperature (65 °C) and retained high activity after prolonged incubation at 45 °C. The reduction of OTA levels in food products by AnOTA has been investigated using several commercial plant-based beverages. The results showed complete degradation of OTA with no detectable modification of beverage proteins. Therefore, the addition of AnOTA seems to be a useful procedure to eliminate OTA in plant-based beverages. Moreover, computational predictions of in vivo characteristics indicated that AnOTA is neither an allergenic nor antigenic protein. All characteristics found for AnOTA supported the suitability of its use for OTA detoxification in food and feed.

The interaction of the azetidine thiazole side chain with the active site loop (ASL) 3 drives the evolution of IMP metallo-β-lactamase against tebipenem.

Imipenemase (IMP) metallo-β-lactamases (MBLs) hydrolyze almost all available β-lactams including carbapenems and are not inhibited by any commercially available β-lactamase inhibitor. Tebipenem (TP) pivoxil is the first orally available carbapenem and possesses a unique bicyclic azetidine thiazole moiety located at the R2 position. TP has potent in vitro activity against Enterobacterales producing extended-spectrum and/or AmpC β-lactamases. Thus far, the activity of TP against IMP-producing strains is understudied. To address this knowledge gap, we explored the structure activity relationships of IMP MBLs by investigating whether IMP-6, IMP-10, IMP-25, and IMP-78 [MBLs with expanded hydrolytic activity against meropenem (MEM)] would demonstrate enhanced activity against TP. Most of the Escherichia coli DH10B strains expressing IMP-1 variants displayed a ≥twofold MIC difference between TP and MEM, while those expressing VIM or NDM variants demonstrated comparable MICs. Catalytic efficiency (kcat/KM) values for the TP hydrolysis by IMP-1, IMP-6, IMP-10, IMP-25, and IMP-78 were significantly lower than those obtained for MEM. Molecular dynamic simulations reveal that V67F and S262G substitutions (found in IMP-78) reposition active site loop 3, ASL-3, to better accommodate the bicyclic azetidine thiazole side chain, allowing microbiological/catalytic activity to approach that of comparison MBLs used in this study. These findings suggest that modifying the R2 side chain of carbapenems can significantly impact hydrolytic stability. Furthermore, changes in conformational dynamics due to single amino acid substitutions should be used to inform drug design of novel carbapenems.

PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity

Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1–3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

Extracellular Domain of IL-10 Receptor Chain-2 (IL-10R2) and Its Arginine-Containing Peptides Are Susceptible Substrates for Human Prostate Kallikrein-2 (KLK2).

The kallikrein-related peptidase KLK2 has restricted expression in the prostate luminal epithelium, and its protein target is unknown. The present work reports the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy-transfer peptides from which the sequence SYRIF was the most susceptible substrate for KLK2. The sequence SYRIF is present at the extracellular N-terminal segment (58SYRIF63Q) of IL-10R2. KLK2 was fully active at pH 8.0-8.2, found only in prostate inflammatory conditions, and strongly activated by sodium citrate and glycosaminoglycans, the quantities and structures controlled by prostate cells. Bone-marrow-derived macrophages (BMDM) have IL-10R2 expressed on the cell surface, which is significantly reduced after KLK2 treatment, as determined by flow cytometry (FACS analysis). The IL-10 inhibition of the inflammatory response to LPS/IFN-γ in BMDM cells due to decreased nitric oxide, TNF-α, and IL-12 p40 levels is significantly reduced upon treatment of these cells with KLK2. Similar experiments with KLK3 did not show these effects. These observations indicate that KLK2 proteolytic activity plays a role in prostate inflammation and makes KLK2 a promising target for prostatitis treatment.

Genome-Wide Isolation of VIN Gene Family and Functional Identification of HpVIN4 in Red Pitaya (Hylocereus polyrhizus)

Soluble sugars, including glucose, fructose and sucrose, are the most important determinants that affect the flavor and quality of red pitaya (Hylocereus polyrhizus) fruit. Vacuolar invertase (VIN), which catalyzes sucrose hydrolysis into glucose and fructose, is a key type of enzyme responsible for soluble sugar metabolism in plant growth and development. Herein, we conducted genome-wide identification, gene expression analysis, subcellular localization and an enzymatic properties assay for the VIN-encoding genes from red pitaya. During red pitaya fruit development towards ripening, the enzymatic activities of VIN showed an up-regulated trend towards ripening. In total, four isoforms (HpVIN1–4) of the VIN-encoding gene were identified from the pitaya genome. Sequence alignment results revealed that the HpVIN1, HpVIN3 and HpVIN4 proteins contained essential motifs for targeting the vacuole and conserved motifs or residues responsible for sucrose binding and hydrolysis. Gene expression pattern analyses revealed that the level of HpVIN4 was obviously increasing during fruit development and acted as the most abundant VIN isoform towards ripening. Subcellular localization detection via transient expression in Arabidopsis thaliana mesophyll protoplasts revealed that the HpVIN4 protein was localized in the vacuole. Growth complementation tests of heterologous expression in the invertase-deficient baker’s yeast strain suggested that the HpVIN4 protein had a sucrose hydrolysis activity and could restore the yeast growth in vivo. The identification of enzymatic properties in vitro demonstrated that the HpVIN4 protein could degrade sucrose into glucose and fructose with an optimum pH of 4.0. Specifically, the HpVIN4 protein had an estimated Km value of 5.15 ± 1.03 mmol·L−1 for sucrose hydrolysis. Ultimately, this study provides a comprehensive understanding of the potential roles of VINs during fruit development and towards ripening and provides functional gene resources for regulating soluble sugar accumulation in red pitaya fruit.

Adsorption of extracellular lipase in a packed-bed reactor: an alternative immobilization approach.

In light of the growing demand for novel biocatalysts and enzyme production methods, this study aimed to evaluate the potential of Aspergillus tubingensis for producing lipase under submerged culture investigating the influence of culture time and inducer treatment. Moreover, this study also investigated conditions for the immobilization of A. tubingensis lipase by physical adsorption on styrene-divinylbenzene beads (Diaion HP-20), for these conditions to be applied to an alternative immobilization system with a packed-bed reactor. Furthermore, A. tubingensis lipase and its immobilized derivative were characterized in terms of their optimal ranges of pH and temperature. A. tubingensis was shown to be a good producer of lipase, obviating the need for inducer addition. The enzyme extract had a hydrolytic activity of 23 U mL-1 and achieved better performance in the pH range of 7.5 to 9.0 and in the temperature range of 20 to 50°C. The proposed immobilization system was effective, yielding an immobilized derivative with enhanced hydrolytic activity (35 U g-1), optimum activity over a broader pH range (5.6 to 8.4), and increased tolerance to high temperatures (40 to 60 ℃). This research represents a first step toward lipase production from A. tubingensis under a submerged culture and the development of an alternative immobilization system with a packed-bed reactor. The proposed system holds promise for saving time and resources in future industrial applications.

An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout.

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

Effect of RE-O/C catalyst on the hydrolysis kinetics and catalytic mechanism of MgH2

The reaction between MgH2 and water produces a significant amount of hydrogen gas, which is beneficial for the development of hydrogen energy in fuel cells. However, the slow hydrogen production rate and the formation of a dense Mg(OH)2 layer limit its application. In this study, MgH2 was doped with 2 wt%, 4 wt%, and 8 wt% of RE-O/C (RE-O = La2O3, CeO2, Pr6O11) catalysts through ball milling technology, and hydrolysis experiments were conducted. The hydrolysis kinetics and activation energy were analyzed using Avrami-Erofeev and Arrhenius equations for linear fitting. X-ray diffraction and scanning electron microscopy were employed to analyze the hydrolysis mechanism and the effect of the catalysts on particle surfaces. Notably, the addition of 4 wt% RE-O/C exhibited the best improvement in the hydrolysis performance of MgH2. At high temperatures, the maximum hydrogen production rates for MgH2+4La2O3/C, MgH2+4CeO2/C, and MgH2+4Pr6O11/C are 32.05, 30.92, and 29 mL·g−1·s−1, respectively, with maximum hydrogen capacities of 1996, 2140, and 2000 mL·g−1, and activation energies of 21.8, 24.8, and 21.3 kJ·mol−1, respectively. The MgH2+RE-O/C composites studied overcame the key issues of MgH2 hydrolysis, presenting a promising strategy for hydrogen production.

Mixed culture fermentation using Lactobacillus delbrueckii and novel Lactiplantibacillus plantarum strains isolated from traditional chhurpi from Sikkim Himalayan region for potential probiotic and improved functional properties

The aim of the study was to isolate potential probiotic lactic acid bacteria (LAB) strains from the traditional fermented milk products, Chhurpi of the Sikkim Himalayan region and to prepare curd using mixed culture fermentation. A preliminary probiotic assessment of 110 LAB isolates including antimicrobial activity, bile and acid tolerance, surface hydrophobicity, auto-aggregation and co-aggregation, and adhesion to Caco-2 cells, was performed. Lactiplantibacillus plantarum strains N4, CHDE2, CHDE4, and SS22 demonstrated better viability in bile (0.3%) and acid (pH 2.5), adherence with hydrophobicity (13.93–92.85%), auto-aggregation (72.44–89.74%), adhesion to Caco-2 cells (45–73.61%) and antimicrobial activity against food borne pathogens. These strains were used for mixed culture fermentation with a previously screened curd forming strain Lactobacillus delbrueckii WS4. Curd produced using mixed culture fermentation of Lb. delbrueckii WS4 and the selected Lp. plantarum strains had a relatively higher acid production (pH 3.97–4.01), protein hydrolysis, and antioxidant activity. Further, mixed culture fermentation also resulted in the increase of HOCl scavenging activity (12.22–32.96%), myeloperoxidase (MPO) inhibition activity (22.38–42.94%) and angiotensin converting enzyme (ACE) inhibition (15.61–33.03%) as compared to that of WS4 fermentation (P < 0.05). This is the first report on identification of potential probiotic strains from the traditional fermented milk products of Sikkim and application in mixed culture fermentation with enhanced multifunctional potential.

Fe3O4@silica-thermolysin: A robust, advantageous, and reusable microbial nanobiocatalyst for proteolysis and milk-clotting

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Physicochemical properties, antioxidant and antidiabetic activities of different hydrolysates of goat milk protein

There is growing interest in the origin, preparation, and application of bioactive peptides. This study investigated the impact of 6 enzymes on the structural, physicochemical properties, antioxidant activities, and antidiabetic potential of defatted fresh goat milk. Structural and functional changes resulting from enzymatic hydrolysis were assessed using gel electrophoresis, laser particle size analysis, multi-spectroscopy, and evaluations of foaming and emulsification properties. Antioxidant capacity was determined through free radical scavenging, Fe2+ chelation, and reducing ability experiments. Additionally, the inhibitory effects of the hydrolysates on α-glucosidase and α-amylase were measured to evaluate antidiabetic activity. Results showed that enzymatic hydrolysis disrupted the spatial structure of goat milk protein and reduced its molecular weight. Papain hydrolysate exhibited the highest degree of hydrolysis (32.87 ± 0.11%) and smallest particle size (294.75 ± 3.33 nm), followed by alcalase hydrolysate (29.12 ± 0.09%, 302.03 ± 7.28 nm). Alcalase hydrolysate showed the best foaming properties, while papain hydrolysate demonstrated the strongest DPPH and hydroxyl radical scavenging activity, Fe2+ chelation, and antidiabetic potential. These findings provide solid theoretical basis for utilizing defatted goat milk as functional ingredients or excipients in the food, medical, and cosmetic industries.

Waterborne atenolol disrupts neurobehavioral and neurochemical responses in adult zebrafish.

Environmental contamination by pharmaceuticals from industrial waste and anthropogenic activities poses adverse health effects on non-target organisms. We evaluated the neurobehavioral and biochemical responses accompanying exposure to ecological relevant concentrations of atenolol (0, 0.1, 1.0, and 10µg/L) for seven uninterrupted days in adult zebrafish (Danio rerio). Atenolol-exposed fish exhibited anxiety-like behavior, characterized by significant bottom-dwelling with marked reduction in vertical exploration. Atenolol-exposed fish exhibited marked increase in the duration and frequency of aggressive events without altering their preference for conspecifics. Biochemical data using brain samples indicated that atenolol disrupted antioxidant enzyme activities and induced oxidative stress. Exposure to atenolol markedly decreased ATP and AMP hydrolysis without affecting ADP hydrolysis and acetylcholinesterase (AChE) activity. Atenolol significantly upregulated tryptophan hydroxylase 1 (tph1) mRNA expression but downregulated brain-derived neurotrophic factor (bdnf) mRNA. Collectively, waterborne atenolol elicits aggressive and anxiety-like responses in adult zebrafish, accompanied by oxidative stress, reduced nucleotide hydrolysis, altered tph1 and bdnf mRNA expression, which may impact the survival and health of fish in aquatic environment.

Allosteric Control of the Catalytic Properties of Dipeptide-Based Supramolecular Assemblies.

Allostery, as seen in extant biology, governs the activity regulation of enzymes through the redistribution of conformational equilibria upon binding an effector. Herein, a minimal design is demonstrated where a dipeptide can exploit dynamic imine linkage to condense with simple aldehydes to access spherical aggregates as catalytically active states, which facilitates an orthogonal reaction due to the closer proximity of catalytic residues (imidazoles). The allosteric site (amine) of the minimal catalyst can concomitantly bind to an inhibitor via a dynamic exchange, which leads to the alternation of the energy landscape of the self-assembled state, resulting in downregulation of catalytic activity. Further, temporal control over allosteric regulation is realized via a feedback-controlled autonomous reaction network that utilizes the hydrolytic activity of the (in)active state as a function of time.