Enzymatic modification of cotton fibre polysaccharides as an enabler of sustainable laundry detergents.

Cotton is the most common natural fibre used in textile manufacture, used alone or with other fibres to create a wide range of fashion clothing and household textiles. Most of these textiles are cleaned using detergents and domestic or commercial washing machines using processes that require many chemicals and large quantities of water and energy. Enzymes can reduce this environmental footprint by enabling effective detergency at reduced temperatures, mostly by directly attacking substrates present in the soils. In the present study, we report the contribution of a cleaning cellulase enzyme based on the family 44 glycoside hydrolase (GH) endo-beta-1,4-glucanase from Paenibacillus polymyxa. The action of this enzyme on textile fibres improves laundry detergent performance in several vectors including soil anti-redeposition, dye transfer inhibition and stain removal. Molecular probes are used to study how this enzyme is targeting both amorphous cellulose and xyloglucan on textile fibres and the relationship between textile surface effects and observed performance benefits.

Spectral modulation of B850 bacteriochlorophyll a in light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum by detergents and calcium ions.

Spectral variations of light-harvesting (LH) proteins of purple photosynthetic bacteria provide insight into the molecular mechanisms underlying spectral tuning of circular bacteriochlorophyll (BChl) arrays, which play crucial roles in photoenergy conversion in these organisms. Here we investigate spectral changes of the Qy band of B850 BChl a in LH2 protein from purple sulfur bacterium Thermochromatium tepidum (tepidum-LH2) by detergents and Ca2+. The tepidum-LH2 solubilized with lauryl dimethylamine N-oxide and n-octyl-ß-D-glucoside (LH2LDAO and LH2OG, respectively) exhibited blue-shift of the B850 Qy band with hypochromism compared with the tepidum-LH2 solubilized with n-dodecyl-ß-D-maltoside (LH2DDM), resulting in the LH3-like spectral features. Resonance Raman spectroscopy indicated that this blue-shift was ascribable to the loss of hydrogen-bonding between the C3-acetyl group in B850 BChl a and the LH2 polypeptides. Ca2+ produced red-shift of the B850 Qy band in LH2LDAO by forming hydrogen-bond for the C3-acetyl group in B850 BChl a, probably due to a change in the microenvironmental structure around B850. Ca2+-induced red-shift was also observed in LH2OG although the B850 acetyl group is still free from hydrogen-bonding. Therefore, the Ca2+-induced B850 red-shift in LH2OG would originate from an electrostatic effect of Ca2+. The current results suggest that the B850 Qy band in tepidum-LH2 is primarily tuned by two mechanisms, namely the hydrogen-bonding of the B850 acetyl group and the electrostatic effect.

Effects of sodium dodecyl sulfate, Sarkosyl and sodium lauroyl glutamate on the structure of proteins monitored by agarose native gel electrophoresis and circular dichroism.

We have studied binding properties of three detergents, i.e., sodium dodecyl sulfate (SDS), Sarkosyl and sodium lauroyl glutamate (SLG), to model proteins based on their effects on electrophoretic mobilities of the proteins using agarose native gel electrophoresis and circular dichroism (CD). This simple technology can evaluate the dissociative properties of bound detergents from the proteins and their effects on protein structure. SDS influenced the electrophoretic mobilities of all model proteins more strongly than the other two detergents, implying a stronger inclination for protein binding and subsequent alterations in protein structure or reductions in activity, which are supported by CD analysis. On the contrary, Sarkosyl and SLG showed weaker binding and interfered less with the structure and biological activities, indicating that these detergents may be useful for protein purification and analysis. It appeared that SLG was weaker in protein binding than Sarkosyl, although the effects of these two detergents appeared to depend on the proteins.

Which detergent is most suitable for the generation of an acellular pancreas bioscaffold?

Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 µm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.

Robust and Irreversible Sortase-Mediated Ligation by Empolyment of Sarkosyl.

Sortase-mediated ligation (SML) is a widely used method for peptide and protein ligation due to ease of substrate preparation and fast enzymatic kinetics. But there are drawbacks that limit broader applications. Sorting motif in substrates may not be exposed to sortase efficiently due to folding or aggregation. In addition, the ligation is reversible under transpeptidation equilibrium that restricts ligation yield. Here we report a simple but robust method to overcome such limitations. By employment of sarkosyl, the detergent alters substrate conformation to raise sorting motif accessibility for sortase catalysis. Moreover, transpeptidation becomes irreversible presumably by formation of micelle to shield ligation products from sortase. In consequence, excellent yields were achieved from sortase variants with different substrate specificity. Notably, this method is compatible with peptides or proteins capable of forming liquid-liquid phase separation (LLPS), presenting a powerful approach for the conjugation of aggregation-prone substrates. Therefore, we believe the sarkosyl-enhanced SML could be widely applied in peptide and protein chemistry and the unique irreversible transpeptidation mechanism offers an insight to detergent-driven equilibrium.

Auto-induction, biochemical characterization and application of a novel thermo-alkaline and detergent-stable lipase (S9 peptidase domain) from Thermotoga petrophila as cleaning additive and degrading oil/fat wastes.

A peptidase S9 prolyl oligopeptidase domain from Thermotoga petrophila RKU-1T (TpS9) was over-expressed as an active, soluble and hyperstable lipolytic enzyme in the mesophilic host system. The sequence analysis demonstrated, TpS9 is an esterase/lipase-like protein belongs to alpha/beta (α/ß)-hydrolase superfamily with a well-conserved penta-peptide (GLSAG) motif and α/ß-hydrolase fold. Various approaches (induction and cultivation) were employed to enrich TpS9 production, 6.04- and 7.26-fold increment was observed with IPTG (0.4 mM) and lactose (200 mM) in the modified 4ZB medium (pH 7.0), but with IPTG-independent auto-induction strategy 9.02-fold augmentation was achieved after 16 h incubation at 24 °C (150 rev min-1). Purified TpS9 showed optimal activity in McIlvaine buffer (pH 6.5) at 80-85 °C, and revealed great thermal (30-85 °C) and pH (6.0-9.0) for 8 h. No obvious constraint was perceived with various metal ions, surfactants, commercial laundry detergents, and chemical modulators. Whereas, TpS9 activity was improved with Ca2+, Mn2+, and Mg2+ by 210 %, 142.5 %, and 134.3 %, respectively. With 2.5 M NaCl (215 %), 50 % (v/v) methanol (140 %), 50 % (v/v) ethanol (126.6 %), 50 % (v/v) n-butanol (122.3 %), 50 % (v/v) isopropanol (120.4 %), 50 % (v/v) acetone (118.6 %) and 50 % (v/v) glycerol (113.2 %) TpS9 activity was also enriched. TpS9 demonstrated great affinity toward natural oils and p-nitrophenyl ester substrates, but showed peak activity with p-nitrophenyl palmitate (3160 U mg-1). Km, Vmax, kcat, Vmax Km-1 and kcat Km-1 of TpS9 with pNPP were 0.421 mM, 4015 µmol mg-1 min-1, 906.4 s-1, 9536.8 min-1, and 2152.96 mM-1 s-1, respectively. Moreover, TPS9 has notable ability to clean stains (5 min) and degrade the animals' fat (3 h). Hence, TpS9 is a favorable candidate as cleaning bio-additive in detergent formulation, fat degradation and various other applications.

Chitosan decorated magnetic nanobiocatalyst of Bacillus derived α-amylase as a role model for starchy wastewater treatment, detergent additive and textile desizer.

In this study, Bacillus tequilensis TB5 α-amylase from rice-milled by-products (rice bran and de-oiled rice bran) was successfully immobilized onto biologically synthesized magnetic nanoparticles fabricated with chitosan (MNP-Ch) and characterized via different biophysical techniques. Furthermore, the study emphasized incorporating this nanostructure framework (MNP@2mgchitosan_DORB-amy and MNP@3mgchitosan_RB-amy) to offer diverse applications, including enzymatic desizing, cleaning starchy stains, and treating synthetic starchy wastewater. An enzyme loading of > 90 % for both enzymes indicated increased binding sites due to the functional moieties of chitosan on the MNP. The Km was 0.28 and 0.31 mg/mL for the immobilized and free forms of DORB-amy, respectively, and 0.18 and 0.27 mg/mL for the immobilized and free forms of RB-amy, respectively. A low Km indicated an increased affinity of MNP-Ch-immobilized forms of enzymes toward the substrate. The performance of both immobilized enzymes improved at a wide range of pH and temperature, which may be attributed to the covalent binding of the enzyme on to the MNP-Ch. The nanobiocatalysts in the detergent act synergistically to fade the starchy stains. Furthermore, an 8-9 TEGEWA scale rating with > 11 % of starch removal was obtained through the biodesizing of starch-sized cotton fabric. The nanobiocatalyst efficiently decomposed starch and liberated 650-670 mg/L of reducing sugar from the synthetic wastewater, therefore offering promising opportunities for its exploration in a wastewater treatment plant. Thus, the study recommends the potential exploration of sturdy matrices like MNP to offer remarkable applications with maximum operational stability, easier recovery, and higher efficiency.

Co-flotation of effluents from detergent and marble processing industries in Denver and dispersed air flotation systems.

Suspended solids in the marble processing wastewater (MPWW) have the potential to pollute receiving media. Likewise, detergent production wastewater (DPWW) needs treatment prior to discharge as they include surfactants and others. Flotation and its modifications are common for separation purposes in various engineering solutions. To increase flotation performance by changing the surface tension some collector and frother chemicals, surfactants are utilized. Detergents are among important surfactants and they may act as both frother and collector in flotation. Therefore, the purpose of this study was to determine the effectiveness of DPWW in co-flotation with MPWW. Two effluents were mixed at varying ratios and dispersed air (DISP) and Denver (DEN) flotation co-treatment were applied to the mixtures. Volume ratio, time and air flow rate on treatment performance were investigated. Turbidity, solids, COD, phosphate removals were achieved at varying levels when the flotation was applied to the mixture. The highest treatment performance was achieved at 90%MPWW-10%DPWW mixture. 10 min flotation time and 2 L min-1 air flow rate for the DEN system, and 20 min and 6 L min-1 for the DISP system were recommended. Under these conditions turbidity, SS, COD, phosphate and alkalinity residuals (and removal efficiencies) were 2400 NTU(82%), 1720 mg.L-1(89%), 313.6 mg.L-1(10%), 20 mg.L-1(20%) and 600 mg.L-1CaCO3(92%) in the DEN system, respectively. Whereas, in the DISP system, under the same conditions, final values of 1880 NTU(86%), 1540 mg.L-1(91%), 262 mg.L-1(17%), 21 mg.L-1(20%) and 470 mg.L-1(94%) were obtained, respectively. The highest SludgeSS concentration increased up to 19300 mg.L-1 in the 90%-10% mixture. In all samples, dewaterable sludge was obtained. By this study, co-flotation of these two effluents was recommended. Within SDGs, this approach will replace frother chemical usage. The process performance can further be enhanced via flotation modifications and technology can be developed as further study.

Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target. OBJECTIVE: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment. METHODS: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied. RESULTS: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ. CONCLUSION: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.

19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.

A Zwitterionic Detergent and Catalyst-Based Single-Cell Proteomics Using a Loss-Free Microhole-Collection Disc.

Recent advances in single-cell proteomics have solved many bottlenecks, such as throughput, sample recovery, and scalability via nanoscale sample handling. In this study, we aimed for a sensitive mass spectrometry (MS) analysis capable of handling single cells with a conventional mass spectrometry workflow without additional equipment. We achieved seamless cell lysis and TMT labeling in a micro-HOLe Disc (microHOLD) by developing a mass-compatible single solution based on a zwitterionic detergent and a catalyst for single-cell lysis and tandem mass tag labeling without a heat incubation step. This method was developed to avoid peptide loss by surface adsorption and buffer or tube changes by collecting tandem mass tag-labeled peptide through microholes placed in the liquid chromatography injection vials in a single solution. We successfully applied the microHOLD single-cell proteomics method for the analysis of proteome reprogramming in hormone-sensitive prostate cells to develop castration-resistant prostate cancer cells. This novel single-cell proteomics method is not limited by cutting-edge nanovolume handling equipment and achieves high throughput and ultrasensitive proteomics analysis of limited samples, such as single cells.

Extending the Lifetime of Frying Oil through Optimization of Fryer Cleaning.

With increases in consumer demand for fried foods in Japan over the last several decades, the consumption of frying oil has also steadily increased. Fryers used in restaurants to cook large quantities of food are typically cleaned using neutral kitchen detergents at the end of the day after removing the oil from the tank. However, significant amounts of debris can remain in the fryer after cleaning, possibly accelerating oil deterioration and thus reducing the quality of the fried foods. In this study, debris obtained from fryer tanks used in actual restaurants was assessed using scanning electron microscopy-energy dispersive X-ray spectroscopy together with Fourier transform infrared spectroscopy, and were determined to comprise polymerized oil and carbonized organic matter. Experiments using artificially prepared debris confirmed that these materials increased the acid value (AV) of frying oil. Trials in two restaurants serving similar amounts of fried chicken, French fries and doughnuts examined the effects of cleaning the fryer with either an alkaline detergent or a neutral kitchen detergent on debris removal and oil life. The alkaline detergent was found to completely remove debris while the neutral detergent left significant amounts of debris. After cleaning, the fryers were operated with new oil as usual and the deterioration of this oil was monitored by assessing the color difference, AV, carbonyl value and peroxide value. These indices increased 1.3 to 2.0 times faster in the case that the neutral kitchen detergent was used, suggesting that cleaning fryer tanks with an alkaline detergent could contribute to extending the lifetime of frying oil, reducing food losses and thus achieving sustainable development goals.

Shotgun proteomics of detergent-solubilized proteins from Trypanosoma evansi.

Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SIGNIFICANCE: Trypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as "surra" in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.

Concentration of Diynoic Acids in Bicellar Mixtures Derived from Those Phase Separation.

Bicellar mixtures containing diacetylene molecules, such as diynoic acids, can be used as parent materials for functional membranes. A bicellar mixture consisting of a diynoic acid-10,12-tricosadiynoic acid (TCDA)-, a phospholipid-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-, and a detergent-3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO)-was evaluated for its morphology and packing of TCDA molecules in its bicellar mixture. A TCDA/DMPC vesicle was prepared at different molar ratios, TCDA/DMPC = 2/8, 5/5, and 8/2; a TCDA/DMPC/CHAPSO bicellar mixture was prepared by mixing a CHAPSO solution with a TCDA/DMPC vesicle solution as a detergent at different composition ratios, x TCDA/DMPC = [TCDA/DMPC]/([TCDA/DMPC]+[CHAPSO]), of 1.0, 0.70, 0.50, and 0.30. A DMPC molecule formed a bilayer membrane structure and was used to suppress its precipitation. The packing density of the TCDA/DMPC/CHAPSO bicellar mixtures was increased by mixing a CHAPSO molecule in x TCDA/DMPC = 1.0 to 0.70 or 0.50. A TEM image of a TCDA/DMPC/CHAPSO bicellar mixture showed many discoidal assemblies at x TCDA/DMPC = 0.5 of TCDA/DMPC = 5/5. Polymerization of the TCDA molecules in the bicellar mixture by UV light suggested an ordered arrangement of TCDA. Polymerization at x TCDA/DMPC = 0.70 and 0.50 correlated with improved packing density.

Metagenomic exploration of cold-active enzymes for detergent applications: Characterization of a novel, cold-active and alkali-stable GH8 endoglucanase from ikaite columns in SW Greenland.

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture-independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate-modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis. Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli, emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase-family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl2, making it a promising candidate for detergent and washing industry applications.

Detergent-resistant α-amylase derived from Anoxybacillus karvacharensis K1 and its production based on whey.

In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg-1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg-1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg-1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5'-dithio-bis-[2-nitrobenzoic acid (DNTB), ß-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme's activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL-1 and 1580.3 ± 183.7 µmol mg-1 protein min-1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with ß-glucosidase for 24 h.

Purification of Potassium Ion Channels Using Styrene-Maleic Acid Copolymers.

Structural studies require the production of target proteins in large quantities and with a high degree of purity. For membrane proteins, the bottleneck in determining their structure is the extraction of the target protein from the cell membranes. A detergent that improperly mimics the hydrophobic environment of the protein of interest can also significantly alter its structure. Recently, using lipodiscs with styrene-maleic acid (SMA), copolymers became a promising strategy for the purification of membrane proteins. Here, we describe in detail the one-step affinity purification of potassium ion channels solubilized in SMA and sample preparation for future structural studies.

Advancing surgical instrument safety: A screen of oxidative and alkaline prion decontaminants using real-time quaking-induced conversion with prion-coated steel beads as surgical instrument mimetic.

Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the establishment of TESSA (sTainlESs steel-bead Seed Amplification assay), a modified real-time quaking induced cyclic amplification (RT-QuIC) assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

Rigid Cyclic Fluorinated Detergents: Fine-Tuning the Hydrophilic-Lipophilic Balance Controls Self-Assembling and Biochemical Properties.

We report herein the synthesis of three detergents bearing a perfluorinated cyclohexyl group connected through a short, hydrogenated spacer (i.e., propyl, butyl, or pentyl) to a ß-maltoside polar head that are, respectively, called FCymal-3, FCymal-4, and FCymal-5. Increasing the length of the spacer decreased the critical micellar concentration (CMC), as demonstrated by surface tension (SFT) and isothermal titration calorimetry (ITC), from 5 mM for FCymal-3 to 0.7 mM for FCymal-5. The morphology of the micelles was studied by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), indicating heterogeneous rod-like shapes. While micelles of FCymal-3 and -4 have similar hydrodynamic diameters of ∼10 nm, those of FCymal-5 were twice as large. We also investigated the ability of the detergents to solubilize lipid membranes made of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC). Molecular modeling indicated that the FCymal detergents generate disorder in lipid bilayers, with FCymal-3 being inserted more deeply into bilayers than FCymal-4 and -5. This was experimentally confirmed using POPC vesicles that were completely solubilized within 2 h with FCymal-3, whereas FCymal-5 required >8 h. A similar trend was noticed for the direct extraction of membrane proteins from E. coli membranes, with FCymal-3 being more potent than FCymal-5. An opposite trend was observed in terms of stabilization of the two model membrane proteins bacteriorhodopsin (bR) and SpNOX. In all three FCymal detergents, bR was stable for at least 2 months with no signs of aggregation. However, while the structural integrity of bR was fully preserved in FCymal-4 and -5, minor bleaching was observed in FCymal-3. Similarly, SpNOX exhibited the least activity in FCymal-3 and the highest activity in FCymal-5. By combining solubilizing and stabilizing potency, FCymal detergents push forward our expectations of the usefulness of fluorinated detergents for handling and investigating membrane proteins.