Recovery Of Enzyme Activity Research Articles

Resistance and resilience of soil biological indicators: A case study with multi-walled carbon nanotube

Soil ecosystem is experiencing stresses due to climate change, and soil inhabitants try to demonstrate their inherent resistance and resilience against those stresses. Application of nanomaterials as agricultural inputs could bring shifts in resistance and resilience patterns of soil microbes and associated enzymes, especially under short-term heat stress. With this background, the impacts of multi-walled carbon nanotube (MWCNT) on the resistance and resilience of soil biological indicators were evaluated. An incubation experiment was conducted with varied MWCNT concentrations (0, 50, 100, 250, and 500 mg kg-1 soil) for 90 d after 24-h heat stress at 48 ± 2 °C to assess the impacts of MWCNT on soil enzyme activities and microbial populations vis-à-vis their resistance and resilience indices under short-term exposure to heat stress. Enzyme activities were reduced after exposure to heat stress. Resistance indices of enzyme activities were enhanced by MWCNT application on day 1 after heat stress, whereas there was no recovery of enzyme activities after 90-d incubation. Like soil enzyme activities, resistance index values of soil microbial populations followed the similar trend and were improved by MWCNT application. Multi-walled carbon nanotube has the potential to improve resistance indices of soil enzyme activities and microbial populations under heat stress, although they could not recover to their original state during periodical incubation after heat stress. This study helps to understand the relative changes of biological indicators under MWCNT and their ability to withstand heat stress.

Radiation-enhanced self-cascade catalytic Ti3C2Tx-based platform enables controlled release of trans-resveratrol for synergistic radiosensitization against metastasis of orthotopic breast cancer

Synergistic radiosensitization of cancer cells by metal-based nanomaterials and natural products is a forward-looking alternative for radiotherapy enhancement while inhibiting tumor metastasis. This work explores a phase change material (PCM)-gated Ti3C2Tx nanosheet as a photothermal-responsive drug delivery system for loading natural trans-resveratrol (RSV), aiming at such synergistic radiosensitization in a controlled manner. This system can improve the biodispersity of RSV in the physiological solutions and inhibit the radiation-enhanced enzymatic activity of Ti3C2Tx nanosheets to a large extent. Upon 808 nm near infrared light illimitation, Ti3C2Tx-mediated photothermal heating makes the loaded PCM to be melted, resulting in the controlled release of RSV and simultaneous recovery of radiation-enhanced enzymatic activity of Ti3C2Tx nanosheets. By further irradiating with X-ray, Ti3C2Tx nanosheets is responsible for enhanced catalytic decomposition of H2O2 to highly toxic hydroxyl radical as well as the consumption of intracellular glutathione. Both in vitro and in vivo studies demonstrate that those excess radical oxygen species can synergize with the released RSV to induce cancer cell death by apoptosis/ferroptosis hybrid pathway, fully inhibiting growth and metastasis of orthotopic breast cancer. These results reveal that the combination of PCM-gated Ti3C2Tx nanosheets with RSV is capable of fabricating the photothermal-responsive theranostic systems and aiding the confinement of synergistic radiosensitization exclusively in the cancer cells for radiotherapy enhancement against tumor metastasis.

Three-phase partitioning for isolating peroxidase from lemon peels

Background and objective: Peroxidase is an oxidoreductase that uses different compounds as substrates and thus can be utilized for different applications. The goal of this work is to isolate peroxidase from lemon peels using Three-Phase Portioning (TPP). Methods: TPP was set by adding varying amounts of salts and alcohol and the enzyme activity recovery was measured for each variable. Different parameters were optimized successively in order to achieve the highest enzyme activity recovery including salt type, salt concentration, pH, alcohol/crude extract ratio and type of alcohol and then, combining all optimized conditions together. Results: Salt that gave maximal recovery was sodium potassium tartrate, optimal salt concentration was 15%, optimal pH was 8, optimal alcohol/crude extract ratio was 1 and t-butanol was preferred to 1-butanol. Efficiently, upon combining all optimized factors, an activity recovery of 175% was obtained. Conclusion: This protocol provides an easy, feasible method to efficiently isolate peroxidase from lemon peels using TPP.

The role of oxidative stress, tumor and inflammatory markers in colorectal cancer patients: A one-year follow-up study

Oxidative stress (OS) and inflammation are known to play an important role in colorectal cancer (CRC). This study analyzed tumor, inflammatory and OS markers in CRC patients and in a control group. In addition, the evolution of these markers was evaluated after one-year of follow-up treatment. This was a longitudinal and prospective, observational study in 80 CRC patients who were candidates for tumor resection surgery and/or chemo-radiotherapy treatment and a healthy control group (n = 60). Subsequently, catalase (CAT), reduced glutathione (GSH), oxidized glutathione (GSSG) and GSSG/GSH ratio in serum and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and F2-IsoProstanes (F2-IsoPs) in urine at 1, 6 and 12 months after treatment was analyzed. Tumor markers (CEA and CA 19.9), as well as inflammatory markers—leukocytes, neutrophils, neutrophil/lymphocyte (N/L) index, platelets, fibrinogen, C-reactive protein (CRP), and interleukin 6 (IL6)— were also analyzed. As expected, levels of CEA and CA 19.9 and markers of inflammation, except CRP, were significantly higher in CRC compared to the control group. Regarding OS markers, a decrease in CAT and GSH and an increase in GSSG, GSSG/GSH ratio, 8-oxodG and F2-IsoPs were found in CRC patients compared to healthy controls at baseline. After treatment, an improvement of their inflammation profile was accompanied by a progressive recovery of antioxidant enzyme activities and the decline of oxidative byproducts both in serum and urine. Based on the results obtained, we propose the assay of urinary 8-oxodG and F2-IsoPs, as well as serum CAT, GSH, GSSG as a marker for the evaluation of OS and the clinical follow-up of CRC patients.

L-asparaginase immobilization in supramolecular nanogels of PEG-grafted poly HPMA and bis(α-cyclodextrin) to enhance pharmacokinetics and lower enzyme antigenicity

L-asparaginase (ASNase) enzyme has limited therapeutic use due to its poor pharmacokinetics and immunogenicity. To overcome these obstacles, we immobilized ASNase in biocompatible poly hydroxypropyl methacrylamide (P(HPMA))-based nanogels simply formed through the host-guest inclusion complex of ASNase-conjugated random copolymer of HPMA and polyethylene glycol (PEG) acrylate (P(HPMA-MPEGA)) and α-cyclodextrin dimer (bisCD) using cystamine as a linker. The effects of bisCD and polymer concentrations on particle size, gelation time, and recovery of enzyme activity were investigated. The ASNase-conjugated bisCD nanogels were discrete, homogeneous, and spherical with a mean projected diameter of 148 ± 41 nm. ASNase immobilized in the bisCD nanogels caused cytotoxicity on HL-60 cell line with IC50 of 3 IU/ml. In-vivo rat study revealed that the immobilized ASNase reduced the enzyme antigenicity and resulted in 8.1 folds longer circulation half-life than the native enzyme. Conclusively, immobilization of ASNase in P(HPMA-MPEGA) and bisCD supramolecular nanogels could enhance the therapeutic value of ASNase in cancer chemotherapy.

Study of stability, kinetic parameters and release of lysozyme immobilized on chitosan microspheres by crosslinking and covalent attachment for cotton fabric functionalization

Chitosan microspheres are used in the biotechnological, biomedical, and enzyme immobilization fields. This study synthesized lysozyme-chitosan microspheres by crosslinking and covalent attachment. The synthesis conditions were adjusted, and spherical particles content lysozyme were obtained and characterized by Scanning electron microscopy (SEM) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Activity and stability assays were conducted to determine the immobilization method's influence, and the kinetics parameters were calculated. The enzymatic activity recovery yields of 65.8% and 20.0% were obtained for crosslinking and covalent attachment, respectively. Surface lysozyme-chitosan microspheres' enzymatic activity was obtained by covalent attachment. By the crosslinking method, surface and into particle enzyme activity were observed. The functionalization of cotton fabric was achieved, and the impregnation slightly modified cotton fabric properties such as resistance and permeability. Finding antimicrobial properties against bacteria S. aureus and P. aeruginosa, with a growth of 49.12% and 65.31%, respectively, were obtained.

Separation and Purification of Recombinant β-Glucosidase with Hydrophobicity and Thermally Responsive Property from Cell Lysis Solution by Foam Separation and Further Purification.

The aim of this study was to separate and purify recombinant β-glucosidase (GLEGB) with elastin-like polypeptide (ELP) and graphene-binding peptide (GB) from cell lysis solution by foam separation and further purification. The study of foam property of GLEGB cell lysis solution indicated that it had excellent foaming property and foam stability, which was suitable for foam separation. This could be due to the GB tag with hydrophobicity, which made the recombinant β-glucosidase with GB preferentially adsorb on the surface of bubbles. At optimum operating conditions of foam separation, the enzyme activity recovery of GLEGB could reach 95.63 ± 1.0%. The foam solution of GLEGB was further purified based on the thermally responsive property of the ELP tag, and the purification fold of GLEGB could reach 29.6 ± 0.5 at the optimum operating conditions. The prominent purification effect indicates that this technique is a simple and efficient technique for the separation and purification of recombinant enzymes.

Design and synthesis of thermosensitive target segment resin with precisely controlled distance between immobilization sites for immobilization of penicillin G acylase

PurposeTo save the economic cost and improve the performance of enterprises, this study aims to synthesize high performance immobilized penicillin G acylase (PGA) carriers with fast reaction speed, high recovery rate of enzyme activity and good reusability through corresponding theoretical guidance and experimental exploration.Design methodology approachA diblock resin was synthesized by reversible addition-fragmentation chain transfer polymerization method using N, N-diethylacrylamide (DEA) and β-hydroxyethyl methacrylate (HEMA) as functional monomers poly(N, N-diethylacrylamide)-b-poly(β-hydroxyethyl methacrylate) (PDEA-b-PHEMA) was obtained, and the effect of the ratio of DEA and HEMA on the activity of PGA was investigated, and the appropriate block ratio of DEA and HEMA was obtained. After that, the competitive rate of HEMA and glycidyl methacrylate (GMA) under the carrier preparation conditions was investigated. Based on the above work, a thermosensitive resin carrier PDEA-b-PHEMA-b-P(HEMA-co-GMA) with different target distances was synthesized, and the chemical structures and molecular weight of copolymers were investigated by hydrogen NMR (1H NMR).FindingsThe lower critical solution temperature of the resin support decreases with the increase of the monomer HEMA in the random copolymerization; the catalytic performance study indicated that the response rate of the immobilized PGA is fast, and the recovery rate of the enzyme activity of the immobilized PGA varies with the distance between the targets. When the molar ratio of HEMA to GMA in the resin block is 8.15:1 [i.e. resin PDEA100-b-PHEMA10-b-P(HEMA65-co-GMA8)], the activity recovery rate of immobilized PGA can reach 50.51%, which was 15.49% higher than that of pure GMA immobilized PGA.Originality valueThis contribution provides a novel carrier for immobilizing PGA. Under the optimal molar ratio, the enzyme activity recovery could be up to 50.51%, which was 15.49% higher than that of PGA immobilized on the carrier with nonregulated distance between two immobilization sites.

Dietary nano-selenium alleviates heat stress-induced intestinal damage through affecting intestinal antioxidant capacity and microbiota in rainbow trout (Oncorhynchus mykiss)

Heat stress-induced intestinal damage is a key event in fish pathology. Nano-selenium (nano-Se) shows remarkably high biological activity and low toxicity, making it an ideal and ecological Se formulation; however, to date, the protective effects of nano-Se against heat stress-induced intestinal injury and pertinent molecular mechanisms remain unknown. Herein, rainbow trout (Oncorhynchus mykiss) were fed either a basal diet or basal diet + 5 mg/kg nano-Se. Samples were collected before (18 °C for 9 days; CG18 and Se18 groups) and after (24 °C for 8 h; CG24 and Se24 groups) heat stress treatment. On heat stress exposure, intestinal villus height, muscularis thickness, and goblet cell number decreased, and expression of tight junction proteins (ZO-1, occludin, and claudin-8d) was downregulated; dietary supplementation with nano-Se alleviated these effects. Furthermore, in the presence of nano-Se, catalase activity was elevated, and expression of diverse heat shock proteins (Hsp70b, Hsp90α, and Hsp30), selenoproteins (Gpx1a, Gpx1b1, and Trx), and anti-inflammatory cytokine (TGF-β) was upregulated. In contrast, nano-Se supplementation significantly alleviated the increase of the expression of pro-inflammatory cytokines (IL-1β and TNF-α) and the malondialdehyde content. We also observed that heat stress markedly increased the relative abundance of Actinobacteria, Firmicutes, Methylobacterium, Akkermansia, and Deinococcus and decreased that of Proteobacteria; nano-Se supplementation restored these changes, making their distribution similar to that in the control group. Overall, our findings suggest that nano-Se plays a protective role against heat stress-induced intestinal damage in rainbow trout by promoting the recovery of antioxidant enzyme activity, enhancing protein repair, alleviating inflammatory responses, and restoring intestinal microbiota composition.

Investigation into the magnetic immobilization of lipase and its application in the synthesis of structured triacylglycerols

In this paper, sn-1,3-specific lipase from Rhizomucor miehei (RML) was immobilized with dialdehyde starch (DAS) as a support activating agent and nano-sized Fe3O4 as a magnetic carrier. The structured triacylglycerol as human milk fat substitutes enriched with OPO was produced. The results showed that the fixed load of lipase was 52.41 mg/g. The enzyme activity recovery was 57.82%. Compared with free lipase, the relative enzyme activity of RML immobilized in magnetic particle was kept at 92% when placing 10 h in a methanol solution. The lipase immobilized in magnetic particle had an average particle diameter of 169 nm and was applied in the synthesis reaction of 1,3-dioleoyl-2-palmitoylglycerol (OPO) by using tripalmitin (PPP) and oleic acid (OA) as raw materials. Under the optimal reaction conditions of 10% immobilized lipase, 5:1 substrate molar ratio (OA/PPP), 50 °C reaction temperature and 7 h reaction time, the product's OPO content was 43.97%, and the content of triacylglycerols rich in palmitic acid at the sn-2 position was 83.32%. After eight repeated uses at 50 °C, the relative activity of RML immobilized in magnetic particle remained above 71%. The synthesized OPO has potential to be used in infant formulas.

Changes in Enzyme Activity in Post-Pyrogenic Soils (Physical Model Experiment)

Presented data on changes in the enzymatic activity of soils under pyrogenic effects in model experiments simulating natural fires. A series of experiments aimed at studying pyrogenic inhibition of enzyme activity and dynamics of their recovery in post-pyrogenic soils were performed under laboratory and natural conditions. The objects of the study were sandy soil (Seropeski, Arenosol) and migratory-segregation black soil (Haplic Chernozem (Loamic, Pachic) Rostov region. Model experiments were carried out by treating soil samples with gas burner flame (amount of heat 87–435 × 103 J) and pyrogenic action of wood fuel combustion (10–279 × 106 J). In the first case, the exposure duration was 1 to 5 min, and in the second case from 1 to 120 min. The response of enzymes (catalase, invertase, urease, peroxidase and phosphatase) to low-temperature plasma exposure (1–120 min) was found to differ depending on the enzyme group and degree of exposure. It was found that the depth of penetration of thermal effects in simulated fires was limited to the top soil layer (0–10 cm), and the radius of spread relative to the combustion source did not exceed 20 cm. Temperature values varied according to time, source of exposure and amount of fuel (400–600°C). Sandy soils and chernozem were heated and cooled down at different rates. The activity of catalase, invertase and urease were found to be inversely related to the intensity of pyrogenic exposure. The dynamics of enzyme activity recovery in post-pyrogenic soils was traced. Invertase activity recovered faster than catalase and urease activity, but full recovery after a year does not occur. The results obtained indicate a significantly prolonged effect of inhibition of enzymatic activity during simulation of natural fires.

Nutrient recovery and changes in enzyme activity during vermicomposting of hydrolysed chicken feather residue

ABSTRACT Chicken feathers are hazardous to the environment because of their poor digestibility and potential as a source of environmental contaminants. However, this waste contains valuable plant nutrients that can be recovered and used to improve soil fertility and agricultural productivity. The objectives of this study were to evaluate how effective vermicomposting is at recovering nutrients and changes in enzymatic activity during vermicomposting of hydrolysed chicken feather residues (HCFR). The study included four treatments with three replications at different HCFR and pelletized wheat straw (PWS) mixing proportions: (T1) 25% HCFR+75% PWS with earthworms, (T2) 25% HCFR+75% PWS without earthworms, (T3) 50% HCFR+50% PWS with earthworms, and (T4) 50% HCFR+50% PWS (w/w) without earthworms. Eisenia andrei was used in the experiment for 120 days. Earthworm treatments recovered more available plant nutrients than non-earthworm treatments by 14% (T1); 50% K (T3); 47% Mg (T3); 75% P (T3); 55% B (T3); 34% Cu (T3); 40% Fe (T1); 46% Mn (T3); 11% Zn (T1). However, was significantly reduced by −80% (T1). Acid phosphatase, arylsulphatase, alanine aminopeptidase, and leucine aminopeptidase were more active in the treatments with earthworms and positively correlated with P and C: N ratio. Alanine aminopeptidase (3752 µmol AMCA.g−1.h−1) and leucine aminopeptidase (4252 µmol AMCL.g−1.h−1) had higher activities in T3 on day 60 of vermicomposting. As a result, the earthworm treatment recovers more plant nutrients than the non-earthworm treatments, and it can be recommended as a better vermicomposting approach for nutrient recovery from HCFR.

Synthesis of polymer-functionalized β-cyclodextrin, Mg2+ doped, coating magnetic Fe3O4 nanoparticle carriers for penicillin G acylase immobilization

Based on the beneficial effects of magnesium ions on biomolecules, it was first introduced into enzyme immobilization carriers to promote the comprehensive performance of immobilized enzymes in repeated use. Firstly, Fe3O4 nanoparticles (NPs) were prepared by the inverse microemulsion method, then, Mg2+ was doped in the process of coating β-cyclodextrin (β-CD) on the surface of Fe3O4 NPs. Afterward, through acetal reaction, glutaraldehyde (GA) was grafted to the material’s surface to obtain Mg2+-Fe3O4 @ β-CD-g-GA NPs, and covalent bond immobilization of Penicillin G acylase (PGA) was realized by the Schiff base reaction of the carriers’ aldehyde group and PGA’s amino group. After optimization of process conditions, the results show that the performances of the immobilized PGA achieve the best, when the concentration of the enzyme solution is 3.00 vol%, pH is 8.0, the immobilization time is 30 h, and the immobilization temperature is 37 °C. Its enzyme activity recovery (EAR), enzyme activity (EA) and enzyme loading capacity (ELC) are 95.6%, 31997 U/g, 110 mg/g, respectively. Lastly, the operational stability, reusability, and storage stability of Mg2+-Fe3O4 @ β-CD-g-GA-PGA NPs were researched. Relative to free PGA, immobilized PGA has better operation stability and storage stability. After 11 repeated uses, the immobilized PGA still has 56% of the initial vitality, and the carrier recovery (Re) is 86.6%.

Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports

The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in β-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M β-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.

Fabrication of polymer functionalized Mn2+-tannic acid coatings on magnetism-responsive nano-microspheres for Immobilized Penicillin G acylase

Firstly, Fe3O4 nanoparticles (Fe3O4 NPs) were synthesized by the inverse microemulsion method, and then, Mn2+ was doped in the process of coating tannin(TA) on the surface of Fe3O4 NPs to obtain Mn2+-Fe3O4@PTA NPs. The composite carrier containing Mn2+ could activate the catalytic activity of immobilized Penicillin G acylase (PGA). After the GMA was introduced into the carrier surface by ring-opening reaction, the above preparation carrier was modified by introducing RAFT reagent to obtain Mn2+-Fe3O4@PTA-GMA-g-RAFT NPs. Lastly, glycidyl methacrylate (GMA) and methyl methacrylate (MMA) were employed as the target monomer and the monomer for regulating the spacing between the targets, respectively. Herein, PGA was covalently immobilized on the surface of the magnetic microspheres by reaction of the amino groups of the PGA molecules with the epoxy groups of the magnetic polymer microspheres. Selections of immobilization conditions, including enzyme concentration, pH, temperature, and time of immobilization, were explored. The condition under which enzyme loading capacity (ELC), enzyme activity (EA), and enzyme activity recovery (EAR) of immobilized PGA were maximized as follows: enzyme concentration 2.5 vol%, pH = 8.0, time 20 h, and temperature 37 °C. Under the optimal immobilization conditions, ELC, EA, and EAR of immobilized PGA were 120 mg/g, 31,361 U/g, and 93.7%, respectively. Immobilized PGA remained 90.1% of initial activity and the carrier recovery was 97.8% after 14 successive cycles. These results indicated that the biocatalytic systems with potential use in biomedical application by the influence of polyhydroxy groups, target spacing, and Mn2+.

Degradation of phenolic inhibitors by laccase immobilized on tannic acid/polyethylenimine modified magnetic nanoparticles

Magnetic Fe3O4 nanoparticles were successfully functionalized with the low-cost mussel-inspired tannic acid (TA)/polyethylenimine (PEI) under mild conditions and then activated with glutaraldehyde (GA) to covalently immobilize laccase for degradation of four model phenolic inhibitors commonly found in lignocellulosic hydrolysate. The immobilized laccase showed the highest relative enzyme activity with the laccase loading of 39.9 mg/g and the enzyme activity recovery of 53.1%. Compared with free laccase, immobilized laccase possessed better relative enzyme activity at acidic pH and wide temperature range, as well as exhibited excellent storage stability and satisfactory reusability. Moreover, it was found that the phenolic degradation products by immobilized laccase did not exhibit obvious inhibition on subsequent enzymatic cellulose hydrolysis. Thus, the low-cost TA/PEI coating for modification of magnetic nanoparticles will provide a promising support for laccase immobilization and the immobilized laccase has great potential in removal of phenolic inhibitors present in lignocellulosic hydrolysate.

Recovery of reduced thiol groups by superoxide-mediated denitrosation of nitrosothiols

Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase.In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O2•−) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40–70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O2•− dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP+-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O2•− indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O2•− or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O2•− appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O2•− could represent a mechanism to regain reducing equivalents in situations of excess O2•− formation, e.g. in the reperfusion phase after ischemia.

The characteristics of protein-glutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application.

Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu2+. The optimum PG substrate was Cbz-Gln-Gly, and the Km and Vmax values were 1.68 mM and 1.41 μM mg protein−1 min−1, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins.

Physiologically-Based Pharmacokinetic Modeling-Guided Dose Management of Oral Anticoagulants when Initiating Nirmatrelvir/Ritonavir (Paxlovid) for COVID-19 Treatment.

Patients with coronavirus disease 2019 (COVID‐19) with cardiovascular diseases who are at higher risk of progressing to critical illness should be treated with nirmatrelvir/ritonavir (Paxlovid). Ritonavir, the booster in nirmatrelvir/ritonavir, modulates multiple drug metabolizing enzymes and transporters, complicating its use in real‐world clinics. We aimed to apply physiologically‐based pharmacokinetic (PBPK) modeling to simulate the complex drug–drug interactions (DDIs) of ritonavir with two anticoagulants, rivaroxaban and racemic warfarin, to address this important clinical conundrum. Simulations were implemented within Simcyp Simulator. Compound and population models were adopted from Simcyp and our previous studies. Upon verification and validation of the PBPK model of ritonavir, prospective DDI simulations with the anticoagulants were performed in both the general population (20–65 years) and geriatric subjects (65–85 years) with or without moderate renal impairment. Elevated rivaroxaban concentrations were simulated with nirmatrelvir/ritonavir treatment, where the impact was more profound among geriatric subjects with renal impairment. The overexposure of rivaroxaban was restored to normal range on day 4 post‐discontinuation of nirmatrelvir/ritonavir, corroborating with the recovery of enzyme activity. A lower 10 mg daily dose of rivaroxaban could effectively maintain acceptable systemic exposure of rivaroxaban during nirmatrelvir/ritonavir treatment. Treatment of ritonavir marginally declined simulated S‐warfarin concentrations, but substantially elevated that of R‐warfarin, resulting in a decrease in the international normalized ratio (INR). As INR only recovered 2 weeks post‐nirmatrelvir/ritonavir treatment, a longer surveillance INR for warfarin becomes important. Our PBPK‐guided simulations evaluated clinically important yet untested DDIs and supports clinical studies to ensure proper anticoagulation management of patients with COVID‐19 with chronic coagulative abnormalities when initiating nirmatrelvir/ritonavir therapy.

Comprehensive proteomic analysis to elucidate the anti-heat stress effects of nano-selenium in rainbow trout (Oncorhynchus mykiss)

Because of the continuous intensification of global warming, extreme climate fluctuations, and high-density farming, cold-water rainbow trout (Oncorhynchus mykiss) are exposed to conditions of heat stress, which has severely impacted their survival and yield. Nano-selenium (nano-Se) shows higher biological activity and lower toxicity and has emerged as an ideal and ecological Se formulation. Herein rainbow trout were fed either a basal diet (control group) or basal diet plus 5 mg/kg nano-Se (treatment group). Samples were collected before (18 °C for 9 days; CG18, Se18) and after (24 °C for 8 h; CG24, Se24) heat stress. The DIA/SWATH approach was then applied to compare changes at the proteome level. We found 223 and 269 differentially abundant proteins in the CG18-CG24 and Se18-Se24 groups, respectively, which mainly included apoptosis-, heat stress-, and lipid-related proteins. In comparison with the CG18-CG24 group, the Se18-Se24 group showed higher abundance of molecular chaperone, such as Hsp70, Hsp90a.1, Hspa8, Hsp30, DNAJA4, Dnajb1, Bag2 and Ahsa1; on nano-Se supplementation, the heat stress-induced decline in the abundance of the selenoprotein MsrB2 was partially restored. Furthermore, nano-Se supplementation downregulated the abundance of lipid-related (CYP51, EBP, DHCR7, DHCR24, and APOB) and pro-apoptotic (caspase-8 and Bad) proteins. Protein–protein interaction analyses suggested that nano-Se inhibits apoptosis by upregulating the expression of Hsp70, Hsp90a.1, Hspa8, and Dnajb1; further, Hsp70/Hspa8 and MsrB2 appear to play a synergistic role in antioxidant defense under heat stress. Overall, our findings provide novel insights into nano-Se-mediated tolerance of heat stress, demonstrating that nano-Se exerts its anti-heat stress effects in rainbow trout by promoting protein repair, enhancing recovery of antioxidant enzyme activity, and alleviating lipid metabolism and apoptosis.