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Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enables highly sensitive detection of HCPs but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 min but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low limit of detection (LOD) and limit of quantification (LOQ) (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in antibody drugs.

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Freezing storage is a common preservation method for industrialized duck meat. However, both the frozen storage and thawing processes of meat can affect meat quality. Therefore, appropriate thawing methods are crucial for maintaining good meat quality. In this study, a pulsed electric field (PEF) was used for thawing zhijiang duck meat and the freshed duck meats were used as control. Optimization of the PEF-assisted thawing process and its effect on the quality of zhijiang duck meat were analyzed. Our data showed that the shear force in the 2 kV/cm PEF-assisted thawing group was the lowest in PEF-assisted thawing groups. The color of zhijiang duck meat in the 2 kV/cm PEF-assisted thawing group was optimal. The 2 kV/cm PEF-assisted thawing could improve the texture characteristics of zhijiang duck meat and enhance water holding capacity of zhijiang duck meat. PEF-assisted thawing could better maintain the microstructure of zhijiang duck meat. Our data showed that if the intensity or duration of PEF treatment is too high, the quality of duck meat will actually decrease. Therefore, appropriate parameters should be selected in practical applications, which will provide a reference for the application of PEF-assisted thawing on the market.

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INTRODUCTION: Yangxinshi tablet (YXST) is a effective traditional Chinese medicine in treating cardiovascular diseases such as heart failure and myocardial infarction. OBJECTIVES: This study aims to develop a method for screening thrombin inhibitors from YXST using an online immobilized enzyme microreactor (IMER) based on capillary electrophoresis (CE). MATERIALS AND METHODS: Thrombin (THR) was immobilized on the capillary's inner wall using polydopamine (PDA). The chromogenic substrate S-2238 was employed to assess thrombin (THR) activity and kinetic parameters. The stability and repeatability of the constructed thrombin-immobilized enzyme microreactor (THR-IMER) were evaluated over 40 runs, maintaining 85% of initial activity. The Michaelis-Menten constant (Km) for THR was determined to be 11.98 mM. The half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) for argatroban on THR were calculated. Ten compounds in YXST were screened for THR inhibitory potency using the THR-IMER. RESULTS: Salvianolic acid B and caffeic acid were identified as potential THR inhibitors in YXST, with inhibition rates at 200 µg/mL of 55.06 ± 6.70% and 31.88 ± 4.79%, respectively, aligning with microplate reader assay results. Molecular docking analysis confirmed their interactions with key THR residues, verifying their inhibitory activity. CONCLUSION: The CE-based THR-IMER method was successfully developed for screening thrombin inhibitors from YXST, offering a reliable approach for identifying potential therapeutic compounds.

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OBJECTIVES: The integration of high-risk human papillomavirus (HPV) detection into cervical cancer screening in Poland aims to improve early detection, yet challenges in coverage and adherence persist. Innovative approaches, like sampling for HPV testing and cytology outside medical settings, have been proposed. This study assesses the feasibility and agreement of results between traditional and novel sampling methods. MATERIAL AND METHODS: A cohort of 50 women aged 25-74 underwent HPV DNA and liquid-based cytology sampling both in-office using standard method and outside the medical setting by trained personnel. Samples were analyzed for HPV DNA using Real-Time PCR and cytology according to the Bethesda System. RESULTS: Cytology and HPV DNA positivity rates showed substantial agreement between methods, with almost perfect agreement for high-risk HPV types. Visual assessment of the cervix was successfully conducted in all cases. Preliminary results suggest remote sampling for HPV DNA and cytology is a viable alternative to traditional methods, with the effectiveness in detecting HPV and cytological abnormalities comparable to this reported in literature, offering potential benefits for individuals with mobility limitations or logistical barriers to attending medical appointments. CONCLUSIONS: The study highlights the potential role of remote sampling for HPV DNA and cytology in enhancing cervical cancer screening accessibility and adherence. Implementation of such methods could improve coverage, particularly among underserved populations. Further research is needed to validate and optimize these approaches for broader clinical use.

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This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes. Proteins can be purified directly from clarified lysate, non-clarified lysate, and even non-lysed cultures without concerns of system clogging. Strong binding between the Ni2+ and the target protein's His6-tag effectively captures the target protein on IMAC resins or membranes placed in the VFD. Continuous flow of different solutions through the VFD allows dynamic binding, washing, and elution of the target protein. Furthermore, the system dramatically accelerates protein purification; a typical purification from cell lysate requires approximately 4 min. Herein, we demonstrate the single-step purification of two His6-tagged proteins from both clarified and non-clarified cell lysates without requiring batch binding. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of the resin-loaded membrane insert and the vortex fluidic device (VFD) setup prior to purification Basic Protocol 2: Purification of His6-tagged proteins using the VFD Alternate Protocol: VFD-mediated His6-tagged protein purification from non-clarified lysate Support Protocol: Preparation of chemically modified glass fiber membrane for VFD-mediated immobilized metal affinity chromatography purification.

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The effect of delignification on the adsorption capacity of loofah sponge-based immobilized metal affinity chromatography adsorbents was investigated with recombinant His-tagged trehalose synthase as the model protein. Pretreatments with [EMIM][Ac] ionic liquid at 80 °C for 5 h and with sodium chlorite/acetic acid at 80 °C for 2 h were found effective for the removal of lignin, leading to a loss in biomass of 15.7% and 25.2%, respectively. Upon delignification, the metal chelating capacities of the loofah sponge-based adsorbents prepared with 5-h ionic liquid pretreatment (712 ± 82 µmole Cu(II)/g) and with 2-h sodium chlorite/acetic acid pretreatment (1012 ± 18 µmole Cu(II)/g) were 38% and 97% higher than that of the control (514 ± 55 µmole Cu(II)/g), adsorbent prepared with untreated loofah sponge, respectively. Results of protein adsorption study indicated that the Co(II)-loaded adsorbent prepared with 2-h sodium chlorite/acetic acid pretreatment exhibited the highest adsorption capacity and selectivity for the recombinant His-tagged trehalose synthase, giving a purification product with a specific activity of 7.62 U/mg protein. The predicted maximum adsorption capacity of the delignified loofah sponge-based adsorbent, 2.04 ± 0.14 mg/g, was 73% higher than that of the control.

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Phthalic acid esters (PAEs) are common hazardous organic contaminants in agricultural soil. Microbial remediation is an effective and eco-friendly method for eliminating PAEs. Nevertheless, the operational mode and potential application of immobilized microorganisms in PAEs-contaminated soil are poorly understood. In this study, we prepared an immobilized bacterial agent (IBA) using a cedar biochar carrier to investigate the removal efficiency of PAEs by IBA in the soil. We found that IBA degraded 88.35% of six optimal-control PAEs, with 99.62% biodegradation of low-molecular-weight PAEs (DMP, DEP, and DBP). The findings demonstrated that the IBA achieved high efficiency and a broad-spectrum in degrading PAEs. High-throughput sequencing revealed that IBA application altered the composition of the soil bacterial community, leading to an increase in the relative abundance of PAEs-degrading bacteria (Rhodococcus). Furthermore, co-occurrence network analysis indicated that IBA promoted microbial interactions within the soil community. This study introduces an efficient method for the sustainable remediation of PAEs-contaminated soil.

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The significant influx of antibiotics into the environment represents ecological risks and threatens human health. Microbial degradation stands as a highly effective method for reducing antibiotic pollution. This study explored the potential of immobilized microbial consortia to efficiently degrade tetracycline. Concurrently, the suitability of different immobilization materials were assessed, with reed charcoal-immobilized consortia exhibiting the highest efficiency in removing tetracycline (92%). Similarly, wheat-bran-loaded bacterial consortia displayed a remarkable 11.43-fold increase in tetracycline removal compared with free consortia. Moreover, adding the carriers increased the nutrients, while the activities of both intracellular and extracellular catalases increased significantly post-immobilization, thus highlighting this enzyme's crucial role in tetracycline degradation. Finally, analysis of the microbial communities revealed the prevalence of Achromobacter and Parapedobacter, signifying their potential as key degraders. Overall, the immobilized consortia not only hold promise for application in the bioremediation of tetracycline-contaminated environment but also provide theoretical underpinnings for environmental remediation by microorganisms.

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The aim of this study was to produce and to characterize craft beer fermented by immobilized yeast cells with the addition of Prokupac grape pomace seed powder (2.5% and 5%), to obtain a beer enriched with phenolic compounds and improved sensory characteristics. The immobilization of the yeast cells was performed by electrostatic extrusion, while the obtained calcium alginate beads were characterized by light and scanning electron microscopy. Phenolic and hop-derived bitter compounds in beer with or without grape pomace seed powder (GS) phenolics were identified using UHPLC Q-ToF MS. The results indicated that GS adjunct significantly shortened the fermentation process of wort and increased the content of phenolic compounds, especially ellagic acid, flavan-3-ols and pro(antho)cyanidins in the final products compared to the control beer. A total of twenty (iso)-α-acids and one prenylflavonoid were identified, although their levels were significantly lower in beers with GS phenolics compared to the control beer. Beers with GS phenolics showed good antioxidant properties as measured by the reduction of ferric ions (FRP) and the scavenging of ABTS•+ and DPPH• radicals. The concentration of immobilized viable yeast cells was higher than 1 × 108 CFU/g wet mass after each fermentation without destroying the beads, indicating that they can be reused for the repeated fermentation of wort. Beers produced with 5% GS added to the wort exhibited the best sensory properties (acidity, astringency, bitterness intensity, mouthfeel, aftertaste and taste), and highest overall acceptability by the panelists. The results showed that grape pomace seed powder present a promising adjunct for the production of innovative craft beer with good sensory properties and improved functionality.

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Oleuropein (OP) is an appreciated compound present not only in fruits but also in leaves of olive trees, which can be transformed into hydroxytyrosol (HT), a substance with high antioxidant activity. In this work, the transformation of an agricultural residue containing OP (olive leaves or wastewater from mills) to the high added value compound HT is accomplished through different enzymatic strategies. Different enzymes were used, immobilized on various supports by diverse binding forces: beta-glucosidase encapsulated in siliceous material, esterases and lipases immobilized on hydrophobic supports (octyl-functionalized amorphous silica and periodic mesoporous organosilica), and esterase immobilized on amine-functionalized ordered mesoporous silica. All these biocatalysts were tested for oleuropein hydrolysis through two different reaction approaches: a) split of glucosidic bond catalyzed by beta-glucosidase (ß-glu), followed by hydrolysis of the aglycon and further ester hydrolysis. 5 mg·mL-1 of ß-glu fully hydrolyzed 5 mM OP at pH 7 and 50°C in 7 days, and further enzymatic hydrolysis of the aglycon yielded near to 0.5 mM HT in the best conditions tested. b) via direct hydrolysis of the ester bond to produce hydroxytyrosol in a one-step reaction using esterases or lipases. The latter reaction pathway catalyzed by lipase from Penicillium camemberti immobilized on octyl-silica (4 mg·mL-1) at 35°C and pH 6 directly produced 6.8 mM HT (1 mg·mL-1), transforming in 12 days near to 30% of the initial 25 mM OP from a commercial olive leaves extract.

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The immobilization of free enzymes is crucial for enhancing their stability in different environments, enabling reusability, and expanding their applications. However, the development of a straightforward immobilization method that offers stability, high efficiency, biocompatibility, and modifiability remains a significant challenge. Silk fibroin (SF) is a good carrier for immobilized enzymes and drugs. Here, we employed urease as a model enzyme and utilized our developed technology called unidirectional nanopore dehydration (UND) to efficiently dehydrate a regenerated SF solution containing urease in a single step, resulting in the preparation of a highly functionalized SF membrane immobilizing urease (UI-SFM). The preparation process of UI-SFM is based on an all-water system, which is mild, green and able to efficiently and stably immobilize urease in the membranes, maintaining 92.7% and 82.8% relative enzyme activity after 30 days of storage in dry and hydrated states, respectively. Additionally, we performed additional post-treatments, including stretching and cross-linking with polyethylene glycol diglycidyl ether (PEGDE), to obtain two more robust immobilized urease membranes (UI-SFMs and UI-SFMc). The thermal and storage stability of these two membranes were significantly improved, and the recovery ratio of enzyme activity reached more than 90%. After 10 repetitions of the enzymatic reaction, the activity recovery of UI-SFMs and UI-SFMc remained at 92% and 88%, respectively. The results suggest that both UND-based and post-treatment-developed membranes exhibit excellent urease immobilization capabilities. Furthermore, the enzyme immobilization method offers a straightforward and versatile approach for efficient and stable enzyme immobilization, while its flexible modifiability caters to diverse application requirements.

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This paper presented a kinetic model of the Langmuir-Hinshelwood-Hougen-Watson (LHHW) type for porous catalysts with simple one-dimensional geometry, including spheres, infinite cylinders, and flat pellets. The model was applied to systems involving immobilized enzymes, where enzymes are attached to porous support materials to enhance stability and reusability. The LHHW model provided a tool for understanding and modeling reaction kinetics in heterogeneous porous catalysts and immobilized enzymes. A nonlinear reaction-diffusion equation was generated using finite-range Fickian diffusion and nonlinear reaction kinetics, crucial for accurately modeling the behavior of immobilized enzymes. This research addressed a gap in the existing literature by introducing fractional derivatives to investigate enzyme reaction kinetics, capturing the complex dynamics of substrate interaction and reaction rates within the porous matrix. An approximation method based on Lucas wavelets was employed to find solutions for substrate concentration and effectiveness factors across various parameter values. The analytical solutions derived from the Lucas wavelet method (LWM) were evaluated against the fourth-order Runge-Kutta method, showing great agreement between the LWM solutions and numerical counterparts. These results optimized diffusion and reaction kinetics, paving the way for advancements in biocatalysis and efficient enzyme reactor design.

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Immobilization of enzymes improves their stability and recoverability and is therefore crucial for scientific research and industrial applications. In this study, phospholipase LM (PLLM) and phospholipase 3G (PL3G) were immobilized using Fe3O4@SiO2@CS-COOH polycarboxylated magnetic nanoparticles (MNPs-COOH) as carriers and then used for degumming soybean crude oil. The immobilization rates and relative enzyme activities of these immobilized phospholipases were evaluated to determine the optimal immobilization parameters. The enzyme activities of PLLM-MNPs-COOH and PL3G-MNPs-COOH were 2830.87 and 1162.25 U/g, respectively. Enzymatic properties of the free and immobilized enzymes were compared. Both immobilized phospholipases exhibited higher condition tolerance and stability after immobilization. After 30-day storage at 4 °C, both immobilized phospholipases retained approximately 1.3 times the residual activity of the corresponding free phospholipases. When the degumming conditions were optimized, the residual phosphorus contents of the PLLM-MNPs-COOH- and PL3G-MNPs-COOH-degummed oils were 4.91 and 7.41 mg/kg, respectively, which were consistent with the safety standards for oil products. After 6 cycles, PLLM-MNPs-COOH and PL3G-MNPs-COOH continued to preserve 71.88 % and 70.00 % of their initial activities, respectively. The immobilized phospholipases are thus suitable for degumming soybean crude oil, and the mixed enzymes exhibited better degumming potential.

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The discovery of pancreatic lipase (PL) inhibitors is an essential route to develop new anti-obesity drugs. In this experiment, chitosan was used to add amino groups to cellulose filter paper (CFP) and then glutaraldehyde was used to covalently combine PL with amino-modified CFP through the Schiff base reaction. Under optimal immobilization conditions, CFP immobilized PL has a wide range of pH and temperature tolerance, as well as excellent reproducibility, reusability and storage stability. Subsequently, 26 natural products (NPs) were screened by immobilized PL with black tea extract having the highest inhibition rate. Three compounds with binding effects on PL (epigallocatechin gallate, theaflavin-3-gallate and theaflavin-3,3'-digallate) were captured. Molecular docking proved that these three compounds have a strong binding affinity for PL. Fluorescence spectra further revealed that theaflavin-3,3'-digallate could statically quench the intrinsic fluorescence of pancreatic lipase. The molecular docking and thermodynamic parameters indicated that electrostatic interaction was considered as the main interaction force between PL and theaflavin-3,3'-digallate. Finally, the potential anti-obesity targets and pathways of the three compounds were discussed through network pharmacology. This study not only proposes a simple and efficient method for screening PL inhibitors, but also sheds light on the anti-obesity mechanism of active compounds in black tea.

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Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins.

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This study investigated the blocking mechanism of immobilized penicillin G acylase (PGA) during the enzymatic synthesis of amoxicillin. Laboratory observations revealed that the primary cause of clogging was the crystallization of the substrate and product on the enzyme surface. Adjusting key parameters can significantly reduce clogging and improve catalytic efficiency. Methanol can decrease enzyme activity, but isopropyl alcohol cleaners can effectively remove clogs and protect enzyme activity. These findings provide an experimental foundation for optimizing the PGA immobilization process, which is crucial for achieving high efficiency and sustainability in industrial production.

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The current study aimed to evaluate the hydrolysis of whole fat milk (WFM) and sweet whey (SW) using ß-galactosidase (ß-gal) after covalent immobilization onto activated alginate/tea waste (Alg/TW) beads as a novel carrier. The optimum temperature for free and Alg/TW/ß-gal was 40 °C and the ideal pH was 7.0. However, Alg/TW/ß-gal displayed better stabilities at high temperatures and a wide pH range. Additionally, the value of Km and Vmax for Alg/TW/ß-gal was higher than the free enzyme. The Alg/TW/ß-gal showed better residual activity (78.6 %) after 90 storage days at 4 °C. The reusability of Alg/TW/ß-gal was very good as it conserved its full activity after 15 consecutive cycles and conserved 93 % of its initial activity after 10 cycles with ONPG (O-nitrophenyl-ß-D-galactopyranoside) and lactose as a substrate, respectively. The impact of Alg/TW/ß-gal on WFM and SW using HPLC analysis revealed a remarkable decrease in lactose concentration and increase of glucose and galactose concentrations. The SW exhibited higher degree of lactose hydrolysis (97.3 %) compared to WFM (62.4 %). Besides, SW had a prominent increase in total phenolic content (96.8 mg/L) compared to WFM (54.3 mg/L). The antioxidant activity had increased after enzyme treatment in both WFM and SW. The GC-MS analysis for volatile compounds identified twenty-five flavour constituents. Finally, Alg/TW/ß-gal has a potential application for obtaining healthy, acceptable, and commercial dairy products of low lactose.

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It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).

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This study investigates the application of crude glycerol to the production of 1,3-propanediol by immobilized cells of Bacillus pumilus. This is a novel application of a naturally occurring producer obtained from a wastewater storage pond in Thailand. Crude glycerol was obtained through the methanolysis of palm oil, which was catalyzed using rice bran lipase. Ten components of the fermentation medium were screened using a Plackett-Burman design. The statistical significance of the results was determined using multiple linear regression with a backward elimination approach. The significance level was set to 5 % (p < 0.05). Only crude glycerol, (NH4)2SO4, MgSO4, and CaCl2 significantly affected 1,3-propanediol production by immobilized B. pumilus. Furthermore, preliminary screenings of environmental conditions used for 1,3-propanediol production were conducted using a Plackett-Burman design. The results showed that the temperature, time, and quantity of immobilized cells were factors that significantly affected 1,3-propanediol yield. Therefore, the quantities of crude glycerol, (NH4)2SO4, MgSO4, and CaCl2 and the temperature, time, and quantity of immobilized cells were optimized using response surface methodology based on a Box-Behnken design. The model predicted a maximum 1,3-propanediol yield of 45.68 g/L with the following conditions: 60 g/L crude glycerol, 5 g/L (NH4)2SO4, 0.55 g/L MgSO4, 0.05 g/L CaCl2, a fermentation duration of 101 h, and a temperature of 25 °C, with 250 g of immobilized cells. The validation trials confirmed a production level of 44.12 ± 1.81 g/L, indicating a 2.86-fold production increase relative to the control group. Overall, this study demonstrates the potential of using crude glycerol as a substrate to improve the yields of 1,3-propanediol produced by B. pumilus.

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While the pH cross-sensitivity of chromoionophore-based ion-selective optodes (ISOs) has often been regarded as a significant limitation, this paper demonstrates how this apparent drawback can be transformed into a beneficial feature. The response range of chromoionophore-based ISOs shifts proportionally with changes in the sample pH. Thus, integrating them with a stable pH gradient across the optode surface, such as those provided by immobilized pH gradient (IPG) gels, allows for significant enhancement of the effective measuring range of chromoionophore-based ISOs while preserving their maximum sensitivity. We show that the measuring range of sodium-selective chromoionophore-based optodes can be increased up to 2.5 log units when used with commercially available IPG gels. This improvement in measuring range is directly correlated with the pH difference in the pH gradient across the optode, suggesting that even greater enhancements are possible with more substantial pH gradients. Furthermore, this approach is not confined to sodium-selective optodes but can be readily adapted to other ion-selective chromoionophore-based optodes, broadening their potential applications and impact in the field of chemical sensing. This work paves the way for the development of more versatile and highly sensitive optodes across a broad range of analytes, leveraging the pH cross-sensitivity as a tool for enhanced performance.