Recovery Of Enzyme Activity Research Articles

Study of the poly (butylene adipate-co-terephthalate)/poly (vinyl alcohol) coated recycled Fe3O4 magnetic particle carriers for immobilization penicillin G acylase

In this work, the process began by coating a layer of poly (butylene adipate-co-terephthalate) (PBAT)/poly (vinyl alcohol) (PVA) on the surface of magnetic Fe3O4 particles (MPs) obtained from the nickel slag. Then, it was grafted by glutaraldehyde (GA) to obtain Fe3O4@PBAT/PVA-g-GA MPs, which were used as a carrier. Finally, the immobilized PGA was achieved by forming a covalent bond through the Schiff base reaction. To confirm each stage, employed Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibration sample magnetometer (VSM), scanning electron microscope-energy spectroscopy of dispersive x-rays (SEM-EDS), inductively coupled plasma mass spectrometry (ICP-MS), and x-ray photoelectron spectrophotometry (XPS). The immobilization conditions were studied and optimized to improve immobilized PGA stability and catalytic activity. The immobilization of PGA demonstrated its optimal performance under the process conditions. The results were achieved using a 2.5 vol.% enzyme solution concentration, a pH of 8.0, an immobilization time of 24 h, and an immobilization temperature of 37 °C. Under these conditions, the immobilized PGA exhibited an enzyme activity recovery (EAR) of 93.71%, an enzyme activity (EA) of 31,367 U/g, and an enzyme loading capacity (ELC) of 111 mg/g. The operating stability, reusability, and storage stability of Fe3O4@PBAT/PVA-g-GA-PGA MPs were investigated. Comparatively, immobilized PGA exhibited superior operational and storage stability compared to free PGA. Even after 11 repeated uses, the immobilized PGA retained 58% of its initial activity, while the carrier recovery (Re) reached 82%. This indicated that the immobilized PGA MPs offer improved longevity and efficiency, making them a promising choice for practical applications.

Magnesium and Zinc as Vital Micronutrients Enhancing Athletic Performance and Recovery – a Review

Introduction and Aim of the ReviewMagnesium and zinc are vital for cellular metabolism and maintaining homeostasis, playing a particularly significant role for athletes. Magnesium, which is largely stored in bones and muscles, is essential for protein synthesis. Zinc, a critical trace element found in muscle and bone tissue, participates in over 300 enzymatic reactions, acts as an antioxidant and making both minerals crucial for physical performance. This study aims to explore the physiological role of magnesium (Mg) and zinc (Zn) in the context of athletic performance and to examine their impact on energy metabolism, muscle function, protein synthesis and potential benefits of Mg and Zn supplementation for improving athletic outcomes and post-exercise recovery. MethodsA comprehensive review of scientific literature was conducted through a thorough search of the PubMed database to explore the latest findings on the utility of magnesium and zinc in sports. Only articles in English were considered. Current KnowledgeThe review results indicate that Mg and Zn play critical roles in energy metabolism, muscle and immune system health. Mg deficiency can impair muscle function and reduce endurance, while its supplementation supports muscle relaxation, cardiovascular health, and respiratory efficiency. Although required in small amounts, Zn is indispensable for enzymatic activity, protein synthesis, hormonal balance, and muscle recovery, making it vital for post-exercise recovery and immune response modulation. ConclusionsAvailable data suggest that Mg and Zn supplementation may positively influence athletic performance, energy metabolism, and recovery post-exercise, though further research is needed to establish specific supplementation guidelines. This review provides a foundation for developing supplementation strategies aimed at enhancing athletic performance and supporting healthy recovery.

Effects of water immersion on immune, intestinal flora and metabolome of Chinese mitten crab (Eriocheir sinensis) after air exposure

Air exposure stress can induce stress response of Eriocheir sinensis and affect its normal life activities. The goal of this study was to investigate the effects of water immersion on the recovery of hepatopancreas immune-related enzyme activity, intestinal microbial diversity and metabolic level of Chinese mitten crabs after exposure to air. The results show that immersion can effectively alleviate the adverse effects of air exposure on the antioxidant capacity and immune capacity of Chinese mitten crabs, and the longer the time of immersion, the more obvious the recovery effect. Among them, the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and acid phosphatase significantly increased after exposure to air (P < 0.05), reached a peak at 3 h, began to decline after immersion, and returned to a level close to the initial value at 24 h (P < 0.05). In addition, after exposure to air, the glucose and total cholesterol in haemolymph of Eriocheir sinensis were significantly different from the initial values (P < 0.05), gradually recovered to the initial level after re-immersion. However, changes in intestinal flora and hepatopancreas metabolism caused by air exposure did not fully recover after water exposure, and its negative effects did not completely disappear. The sequencing results showed that the species composition and diversity of intestinal microorganisms of Chinese mitten crab changed after air exposure and immersion treatment. The relative abundance of Actinomycetes increased significantly, while that of Proteobacteria and Firmicutes decreased significantly. Metabolomics analysis showed that air exposure and immersion destroyed the metabolic balance of amino acids and carnitine, reduced the level of carnitine metabolism, hindered the absorption of nutrients, and led to the accumulation of harmful substances.

A-111 “Activation” Of macro-ast by pyridoxal phosphate supplementation

Abstract Background Type 1 macro-AST is a high molecular weight AST (Aspartate Aminotransferase) formed between an AST monomer and immunoglobulins in plasma, which can cause elevated AST values. It is believed that reduced clearance or excretion of macro-AST can cause elevated AST results. Here we report a case of macro-AST that produced high AST values only by the AST assay that includes pyridoxal phosphate (PLP), not by the one without PLP. Case description A 54 year-old male patient diagnosed with disseminated Bartonellosis was monitored with ‘liver function” tests on a monthly basis, with AST varying between 24 to 73 U/L (Reference interval, 13-47 U/L). The patient’s AST value suddenly jumped to 378 U/L when the laboratory switched to an AST assay supplemented with PLP (named ASTPM) on Roche Cobas c702 analyzer (c702). When the same patient sample was tested using the previous AST assay which does not include PLP, a result of 14 U/L was obtained. Macro-AST was suspected, as all other “liver function” tests were within reference interval. Methods Protein-A and Protein-G were used to remove immunoglobulins from the patient sample and another sample with matching AST as a control, respectively. Three hundred microliters of washed Protein-A or Protein-G Sepharose bead slurry was mixed with 300 microliters of each sample, respectively. Each mixture was incubated at 37oC for 3 hours on a rocker. After centrifugation, the supernatant was transferred to a new tube. AST, ASTPM, and all other tests were performed on c702, and results corrected for dilution. The recovery of enzyme activity or immunoglobulin concentration after the removal of immunoglobulins was calculated, respectively. Results In the original sample, the ratio of ASTPM to AST was 28 (389 U/L vs. 14 U/L) for the patient, and 1.1 (304 U/L vs. 275 U/L) for the control. After the complete removal of immunoglobulin G (IgG), the recovery of ASTPM for the patient was 3% and for the control was 74%. The recovery of ALTPM (supplemented with PLP) was 89% for the patient and 85% for the control, showing the expected loss of enzymatic activity by Protein-A or Protein-G treatment. Conclusions The significant reduction of ASTPM activity (recovery of 3%) after removal of IgG in the patient sample proved that the AST enzyme was associated with IgG. The slight increase of ASTPM activity compared to AST (ratio of 1.1) in the control sample was an expected effect of supplementing PLP in the AST assay, however, the huge increase of ASTPM activity (ASTPM/AST ratio of 28) in the patient sample suggests that PLP “activated” the macro-AST and produced high AST result. This is a concerning effect of supplementing PLP in AST assays because it can cause spurious AST elevations in cases of macro-AST. As more laboratories begin to employ the PLP supplemented aminotransferase assays, it is important to acknowledge the interactions between PLP and macro-AST and the role of the laboratory to identify macro-AST in order to avoid unnecessary, costly, and time-consuming testing and procedures for these patients.

Preparation and recovery performance of modified cellulase with pH-sensitive separation properties

The efficient utilization of biomass resources holds significant importance for sustainable development in the energy, materials, and chemical industries. Therefore, it has garnered widespread attention. In this study, we have successfully achieved pH-sensitive separation and recycling of cellulase in an aqueous system. Firstly, the cellulase was modified by introducing a polyethylene glycol modifier with an aromatic aldehyde group. This modification altered the conformation of the cellulase and created a homogeneous-like system due to the hydrophilicity of the ethoxyl group in the modifier. As a result, the accessibility of the enzyme to the substrate was improved, which led to an increase in the enzyme activity of 116.12 %. The aromatic aldehyde group in the modifier and the primary amino group in the polyethyleneimine (PEI) formed a dynamic Schiff base covalent bond, which had pH-sensitive properties to ensure the completion of the recycling of the enzyme in the aqueous system. It was found that the molecular weight of PEI affected the recovery properties of the enzyme. Under the condition that the enzymatic activity was not damaged, the recovery rate of enzymatic activity increased as the molecular weight of the PEI increased. Particularly, when the molecular weight of PEI was 1800, the modified cellulase still retained an enzymatic activity recovery rate of 80 % after 5 cycles. This research provides a novel solution to improve the efficient hydrolysis of cellulose and the recovery of cellulase.

Enhanced bioremediation of soils contaminated with nicosulfuron using the bacterial complex A12.

To construct an efficient bacterial complex to degrade nicosulfuron and clarify its degradative characteristics, promote the growth of maize (Zea mays), and provide a theoretical foundation for the efficient remediation of soil contaminated with nicosulfuron. Biocompatibility was determined by the filter paper sheet method by mixing Serratia marcescens A1 and Bacillus cereus A2 in a 1:1 ratio, yielding A12. The optimum culture conditions for the bacterial composite were obtained based on a three-factor, three-level analysis using response surface methodology, with 29.25g l-1 for maltodextrin, 10.04g l-1 for yeast extract, and 19.93g l-1 for NaCl, which resulted in 92.42% degradation at 4 d. The degradation characteristics of A12 were clarified as follows: temperature 30°C, pH 7, initial concentration of nicosulfuron 20mg l-1, and 4% inoculum. The ability to promote growth was determined by measuring the ratio of the lysosphere diameter (D) to the colony diameter (d), and the ability of the complex A12 to promote growth was higher than that of the two single strains. Nicosulfuron degradation in sterilized and unsterilized soils reached 85.4% and 91.2% within 28 d, respectively. The ability of the strains to colonize the soil was determined by extraction of total soil DNA, primer design, and gel electrophoresis. The bioremediation effect of A12 was confirmed by the maximum recovery of fresh weight (124.35%) of nicosulfuron-sensitive crop plants and the significant recovery of soil enzyme activities, as measured by the physiological indices in the sensitive plants.

Synthesis and application of mesoporous MIL-88A for enhanced lipase immobilization and high value oil conversion

MIL-88A, a metal–organic framework (MOF), has significant promise for lipase immobilization due to its robust acid and water tolerance coupled with cost-effectiveness. Nonetheless, enhancing the dimensional compatibility of carrier with lipase and substrate is imperative for its broader application. In this study, we innovatively synthesized mesoporous MIL-88A (Meso-MIL-88A) with a fairly wide pore distribution around 10 nm using a soft-template method followed by a citrate dissociation strategy. The adsorption isotherm of Meso-MIL-88A conformed to the Langmuir model, exhibiting a notable increase in the adsorption capacity of lipase ET 2.0 compared to common MIL-88A (C-MIL-88A), attributed to the augmented external specific surface area (59 to 19 m2/g). Moreover, the immobilized lipase on Meso-MIL-88A showcased superior specific activity and enzyme activity recovery in contrast to C-MIL-88A, owing to enhanced structural preservation of lipase protein and reduced mass transfer resistance. Greater stability towards pH and temperature and better dynamic performance of immobilized lipase compared to free lipases exhibited the great advantages of Meso-MIL-88A as immobilized carrier. Notably, in the synthesis of phosphatidyl DHA (docosahexaenoic acid), immobilized CalB (Candida antarctica lipase B) on Meso-MIL-88A exhibited a 41.89 % incorporation of DHA into PC (phosphatidylcholine) within 48 h and an 82.60 % recovery after five batches of use. SEM observations confirmed the sustained integrity of the immobilized lipase structure post-recycling, underscoring the good stability of Meso-MIL-88A in the catalysis system. This study unveils the substantial potential of Meso-MIL-88A in lipase immobilization and offers a facile approach to tailoring the pore size of MOFs for practical application system.

Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

Construction of Immobilized Laccase System Based on ZnO and Degradation of Mesotrione.

Mesotrione (MES) is a new environmental pollutant. Some reports have indicated that microbial enzymes could be utilized for MES degradation. Laccase is a green biocatalyst whose potential use in environmental pollutant detoxification has been considered limited due to its poor stability and reusability. However, these issues may be addressed using enzyme immobilization. In the present study, we sought to optimize conditions for laccase immobilization, to analyze and characterize the characteristics of the immobilized laccase, and to compare its enzymatic properties to those of free laccase. In addition, we studied the ability of laccase to degrade MES, and analyzed the metabolic pathway of MES degradation by immobilized laccase. The results demonstrated that granular zinc oxide material (G-ZnO) was successfully used as the carrier for immobilization. G-ZnO@Lac demonstrated the highest recovery of enzyme activity and exhibited significantly improved stability compared with free laccase. Storage stability was also significantly improved, with the relative enzyme activity of G-ZnO@Lac remaining at about 54% after 28 days of storage (compared with only 12% for free laccase). The optimal conditions for the degradation of MES by G-ZnO@Lac were found to be 10 mg, 6 h, 30 °C, and pH 4; under these conditions, a degradation rate of 73.25% was attained. The findings of this study provide a theoretical reference for the laccase treatment of 4-hy-droxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide contamination.

Abstract 2061: A proven activity-based workflow for the identification and characterization of time-dependent kinase inhibitors using a continuous assay format

Abstract Time-dependent inhibitors (TDIs) of enzyme targets offer distinct advantages for the development of potent and selective compounds with favorable pharmacokinetic and pharmacodynamic properties. Such inhibitors are characterized by non-linear progress curves: after an initial inhibited velocity, a rate constant governs the transition to a final steady-state reaction rate of the inhibited enzyme. A final rate of zero indicates irreversible inhibition, whereas a non-zero final rate indicates slow-binding inhibition. Characterizing these inhibitory modes of action is enabled with a continuous assay format that avoids the common pitfalls and misleading results seen with end-point assays. A continuous assay format enables efficient and robust determination of the kinetic parameters required to drive structure-activity relationship optimization to streamline the development of more effective drugs. It is important to note that simple IC50s for TDIs will not suffice, and can, indeed, also be misleading. We have developed a robust three-step workflow based on kinetic catalytic activity measurements to quickly identify and characterize TDIs. First, dose-response experiments are conducted with and without an enzyme-inhibitor preincubation step. The curvature of the reaction progress curve in the non-preincubated experiment and a shift in IC50 from the preincubated experiment are indicative of TDI. In the absence of TDI, simple IC50s are reported with, if possible, Ki values. If TDI is present, a second experiment is conducted to assess compound reversibility using either a jump-dilution protocol or a novel free-compound clearance method that uses gel filtration spin columns or spin plates. In either protocol, forward progress curve analysis is used to monitor the recovery of enzymatic activity after dilution of inhibitor in solution. Lastly, the potency of the inhibitor is evaluated using kinetic experiments tailored to the nature of the inhibition – either reversible or irreversible. If reversible, then the rate constant from the reversibility experiment is used to determine the residence time of the molecule. If irreversible, then a 24-point dose-response experiment with serial 1.5-fold dilutions is performed, and all the progress curves are globally fit to determine kinact/KI, and, if possible, kinact and KI separately. The method will be fully described through the characterization of known EGFR inhibitors of three inhibition types: fast-off (Gefitinib), slow binding (Lapatinib), and irreversible (Osimertinib). Citation Format: Earl May, Daniel Urul, Khanh Huynh, Susan Cornell-Kennon, Venkatesh Nemmara, Zhibing Lu, Samuel Hoare, Michelle Lyles, Erik Schaefer. A proven activity-based workflow for the identification and characterization of time-dependent kinase inhibitors using a continuous assay format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2061.

6-Hydroxy-2,2,4-trimethyl-1,2,3,4-tetrahydroquinoline Demonstrates Neuroprotective Properties in Experimental Parkinson's Disease by Enhancing the Antioxidant System, Normalising Chaperone Activity and Suppressing Apoptosis.

Parkinson's disease (PD) is a neurodegenerative disease, whereby disturbances within the antioxidant defence system, increased aggregation of proteins, and activation of neuronal apoptosis all have a crucial role in the pathogenesis. In this context, exploring the neuroprotective capabilities of compounds that sustain the effectiveness of cellular defence systems in neurodegenerative disorders is worthwhile. During this study, we assessed how 6-hydroxy-2,2,4-trimethyl-1,2,3,4-tetrahydroquinoline (HTHQ), which has antioxidant properties, affects the functioning of the antioxidant system, the activity of NADPH-generating enzymes and chaperones, and the level of apoptotic processes in rats with rotenone-induced PD. Six groups of animals were formed for our experiment, each with 12 animals. These were: a control group, animals with rotenone-induced PD, rats with PD given HTHQ at a dose of 50mg/kg, rats with PD given HTHQ at a dose of 25mg/kg, animals with pathology who were administered a comparison drug rasagiline, and control animals who were administered HTHQ at a dose of 50mg/kg. The study results indicate that administering HTHQ led to a significant decrease in oxidative stress in PD rats. The enhanced redox status in animal tissues was linked with the recovery of antioxidant enzyme activities and NADPH-generating enzyme function, as well as an upsurge in the mRNA expression levels of antioxidant genes and factors Nrf2 and Foxo1. Administering HTHQ to rats with PD normalized the chaperone-like activity and mRNA levels of heat shock protein 70. Rats treated with the compound displayed lower apoptosis intensity when compared to animals with pathology. Therefore, owing to its antioxidant properties, HTHQ demonstrated a beneficial impact on the antioxidant system, resulting in decreased requirements for chaperone activation and the inhibition of apoptosis processes triggered in PD. HTHQ at a dose of 50mg/kg had a greater impact on the majority of the examined variables compared to rasagiline.

A comparison of dual-enzyme immobilization by magnetic nanoparticles and magnetic enzyme aggregates for cascade enzyme reactions

Immobilized enzymes have been widely used for their efficiency, economy, and environment friendly. In this case, a simpler and effective immobilization method has been developed. In this study, the enzyme immobilized by magnetic beads with genipin (MGE) was prepared by mixing genipin-modified nano-Fe3O4 with (R)− 1-phenylethanol dehydrogenase ((R)-PEDH) and recombinant glucose dehydrogenase (rGDH), and the magnetic cross-linked enzyme aggregates (MCLEAs) was prepared by stirring a mixture of the sulfur precipitate of the (R)-PEDH and rGDH, poly-L-lysine, and cross-linkers. When o-phthalaldehyde (o-PA) was used as a cross-linking agent for the MCLEAs preparation, a protein loading of 99.9% and 62.93% recovery activity of (R)-PEDH and 70.12% of rGDH were obtained. Compared to the MGE, the MCLEAs possessed better enzyme activity recovery. Furthermore, this study tried the synthesis of (R)− 1-(furan-2-yl) ethanol by the immobilized enzymes and obtained a concentration of 1.10 mM for the MCLEAs and 0.24 mM for the MGE at 48 h. This study explored a facile method for co-immobilization of (R)-PEDH and rGDH to synthesize (R)− 1-(furan-2-yl) ethanol.

Acute suppression of translation by hyperthermia enhances anti-myeloma activity of carfilzomib.

Hyperthermia is a unique treatment option for cancers. Multiple myeloma (MM) remains incurable and innovative therapeutic options are needed. We investigated the efficacy of hyperthermia and carfilzomib in combination against MM cells. Although MM cell lines exhibited different susceptibilities to pulsatile carfilzomib treatment, mild hyperthermia at 43℃ induced MM cell death in all cell lines in a time-dependent manner. Hyperthermia and carfilzomib cooperatively induced MM cell death even under suboptimal conditions. The pro-survival mediators PIM2 and NRF2 accumulated in MM cells due to inhibition of their proteasomal degradation by carfilzomib; however, hyperthermia acutely suppressed translation in parallel with phosphorylation of eIF2α to reduce these proteins in MM cells. Recovery of β5 subunit enzymatic activity from its immediate inhibition by carfilzomib was observed at 24h in carfilzomib-insusceptible KMS-11, OPM-2, and RPMI8226 cells, but not in carfilzomib-sensitive MM.1S cells. However, heat treatment suppressed the recovery of β5 subunit activity in these carfilzomib-insusceptible cells. Therefore, hyperthermia re-sensitized MM cells to carfilzomib. Our results support the treatment of MM with hyperthermia in combination with carfilzomib. Further research is warranted on hyperthermia for drug-resistant extramedullary plasmacytoma.

Preparation of amino-functionalized polyethylene-silica composite membrane and FDH immobilization

Enzymatic CO2 conversion represents a highly efficient, energy-conserving, and environmentally sustainable approach to mitigating the increasing pressure caused by huge carbon emissions and generating valuable products. However, several challenges associate with the process including the instability and recovery difficulty of free enzyme, as well as low solubility of CO2 in mild conditions. Immobilizing enzyme on the amino-functionalized supports presents a viable solution to these issues. Considering the wide application of polymer hollow fiber membranes in the field of CO2 gas–liquid membrane contactor and the characteristics of hydrophilic, biocompatible, and high specific surface area of silica nanoparticles, the amino-functionalized polyethylene (PE)-silica (APS) composite membranes were fabricated via different routes. The morphologies, the loading rates of modified SiO2 and reusability of APS membranes were characterized for a crude screening. Following that, seven kinds of APS membranes were used as the carriers of formate dehydrogenase (FDH) that could catalyze the reduction of CO2 to formate, and their immobilization efficiency and catalytic performance were compared. The results show that 1# and 2# membranes based on carboxyl-modified PE with a loading rate of 5.5 %∼5.6 % and the reusability of above 99 % were more ideal carriers than others. The enzyme activity recovery rate of APS-immobilized FDH (FDH-APS) was separately 367 % and 256 %, proving the intensification action of carriers on the enzymatic reaction. After 10 cycles of use, the relative activity of two kinds of FDH-APS was around 70 % and 78 %, much higher than corresponding particles or membrane attached FDH (25.3 % and 20.4 %). After storage at 4 °C for 20 days, the relative activity of FDH-APS was 82.7 % and 81.0 %, while it was only 61.4 % for free enzyme.

Immobilization of Phospholipase D for Production of Phosphatidylserine via Enzyme-Inorganic Hybrid Nanoflower Strategy

Phosphatidylserine (PS) is a natural phospholipid with particular importance in the food, cosmetic, and pharmaceutical industries. Recently, the synthesis of PS mediated by phospholipase D (PLD) has drawn great attention. But the application of free PLD is limited by various drawbacks, including its instability under extreme conditions, difficulties in reuse and recovery, and high costs. In this work, saPLD-inorganic hybrid nanoflowers (saPLD@NFs) were synthesized with PLD from Streptomyces antibioticus (saPLD) as the organic component and Ca3(PO4)2 as the inorganic component. The saPLD@NFs demonstrated outstanding immobilization capability and achieved a 119% enzyme activity recovery rate. Furthermore, the saPLD@NFs exhibited better thermostability and pH stability in comparison to free saPLD. The PS yield of saPLD@NFs was about 57.4% in the first cycles and still reached 60.4% of its initial PS yield after four cycles. After 25 d storage at 4 °C, saPLD@NFs retained 66.5% of its original activity, but free saPLD only retained 38.3%, indicating that saPLD@NFs have excellent storage stability. Thus, this study established a new method of preparing PLD nanoflowers for effective PS synthesis, which might accelerate the practical utilization of this biocatalyst.

Preparation of cross-linked chitosan magnetic microspheres and immobilization of pectinase

Abstract In this paper, the kinetic model under the influence of each factor is given by investigating the effects of distribution effect, external diffusion effect and internal diffusion effect on the immobilized pectinase reaction system. The magnetic chitosan microspheres were prepared by the reversed-phase suspension cross-linking method, and the effects of the type and amount of cross-linking agent and the ratio of chitosan to magnetic core incorporation on the performance of the magnetic spheres were investigated. Then, the immobilized pectinase was prepared by using cross-linked chitosan magnetic microspheres as a carrier, and an orthogonal test was used to determine the conditions of immobilized enzyme preparation and to study the enzymatic properties and operational stability of immobilized pectinase. The results showed that the transmittance of the magnetic microspheres of this test increased from 32% to 90.79% in 10 min in a magnetic field, but in a gravity field, the transmittance increased from 32.12% to 46.44% in 50 min. The experimentally prepared microspheres exhibit good magnetic responsiveness. The optimum temperature of immobilized pectinase was 50°C, and the remaining enzyme activity was still 61.24% after 6 repetitions. The immobilized pectinase made by crosslinking glutaraldehyde with chitosan magnetic microspheres has a high recovery of enzyme activity and good operational stability, as indicated by this.

Assessment of Cu(II) impact on aerobic sludge biomass and its post-exposure self-recovery potential

Heavy metals are released from various sources, including metal plating, potentially at a small-scale industrial level, and from automobile service and repair workshops, paint works, etc., spread across the city. Being part and parcel of the city, the wastewater from these metallic sources finds its way into the sewage system and can potentially disrupt biological sewage treatment plants. Therefore, conducting health and quality assessments of the sludge biomass becomes crucial to ensure the effective operation of these treatment plants and to maintain environmental and public health standards. In this study, impacts of Cu(II) (10, 20, 40, and 80 mg/L) exposure on sludge biomass have been investigated, and the potential of self-recovery of sludge biomass was explored when Cu(II) exposure is stopped. Reactors have been operated for 0–25 d with Cu(II) (termed as stressed phase) and then another 29 d without Cu(II) (termed as recovery phase) in the feed. During stressed phase operation, Cu(II) stressed reactors have shown 39–75 % chemical oxygen demand (COD) removal, compared to 91 % for control. Biomass activity and ECatalase activity have been inhibited under Cu(II) stress with changes in microbial community structure. During the recovery phase, Cu(II) stressed reactors have shown 41–82 % COD removal, whereas the control reactor has shown 91 % COD removal. A low sludge volume index (SVI) and high biomass interface settling velocity (ISV) with increased biomass activity have been obtained for biomass exposed to 10 and 20 mg/L of Cu(II). Lower Cu(II) doses have led to partial recovery of enzyme activity and microbial community structure, but higher doses have shown incomplete recovery. The study shows the impact caused by Cu(II) metal in a biological wastewater treatment reactor. Through comprehensive analysis of basic and advanced physico-chemical and biological parameters, the study recommends preliminary and confirmatory tests to know metallic ingress through wastewater influent in a treatment system. Advanced knowledge about metallic ingress will be useful for operators to take necessary steps for the sustainable operation of wastewater treatment systems.

Novel co-immobilization of thermostable isomerase and aspartase on NH2-UiO66 for efficient biosynthesis of L-aspartate from maleate

L-aspartate is an important chemical in the food and pharmaceutical industries. Presently, it is meaningful to realize a co-immobilized multi-enzyme biocatalyst that can synthesize L-aspartate in vitro through multi-step cascade reaction. Herein, the immobilization method was first employed to investigate the ability of immobilized double enzymes, isomerase AaMaiA and aspartase AspB, to systhesize L-aspartate. The optimal temperature (50 ℃) of AaMaiA-immobilized NH2-UiO66, a metal organic framework material, was significantly superior to that of its free counterpart. Compared to free enzymes, AaMaiA@NH2-UiO66 retained 57.8% of enzyme activity and exhibited a half-life that was improved 2.59-fold at 50 ℃. The co-immobilized AaMaiA/AspB@NH2-UiO66 (AaMaiA: AspB = 6: 1, v/v) showed high protein loading (27.899 ± 1.945 mg/g) and 84.4% recovery of enzyme activity after 8 consecutive cycles. Compared to free AaMaiA/AspB, the novel AaMaiA/AspB@NH2-UiO66 exhibited far superior tolerance to organic solvents. For the first time, the prepared AaMaiA/AspB@NH2-UiO66 was successfully applied as an efficient biocatalyst and converted 1 M maleate to L-aspartate with a yield of 86.8% within 1 h. Remarkably, AaMaiA/AspB@NH2-UiO66 exhibited great potential for efficient biosynthesis of L-aspartate.

Co-encapsulating Cofactor and Enzymes in Hydrogen-Bonded Organic Frameworks for Multienzyme Cascade Reactions with Cofactor Recycling.

Use of hydrogen-bonded organic frameworks (HOFs) for enzyme immobilization faces challenges in the improvement of enzyme activity recovery and the assembly of cofactor-dependent multienzyme systems. Herein, we report a polyelectrolyte-assisted encapsulation approach (PAEA) that enables two cascades with four oxidoreductases and two nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) cofactors co-encapsulated in BioHOF-1 with excellent cargo loading and over 100 % cascade activity. The key role of the polyelectrolyte is to coat enzymes and tether NAD(P)H, thus interacting with HOF monomers in place of enzymes, avoiding the destruction of enzymes by HOF monomers. The versatility and efficiency of PAEA are further illustrated by an HOF-101-based bio-nanoreactor. Moreover, the immobilization by PAEA makes enzymes and NAD(P)H display excellent stability and recyclability. This study has demonstrated a facile and versatile PAEA for fabricating cofactor-dependent multienzyme cascade nanoreactors with HOFs.

Co‐encapsulating Cofactor and Enzymes in Hydrogen‐Bonded Organic Frameworks for Multienzyme Cascade Reactions with Cofactor Recycling

AbstractUse of hydrogen‐bonded organic frameworks (HOFs) for enzyme immobilization faces challenges in the improvement of enzyme activity recovery and the assembly of cofactor‐dependent multienzyme systems. Herein, we report a polyelectrolyte‐assisted encapsulation approach (PAEA) that enables two cascades with four oxidoreductases and two nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) cofactors co‐encapsulated in BioHOF‐1 with excellent cargo loading and over 100 % cascade activity. The key role of the polyelectrolyte is to coat enzymes and tether NAD(P)H, thus interacting with HOF monomers in place of enzymes, avoiding the destruction of enzymes by HOF monomers. The versatility and efficiency of PAEA are further illustrated by an HOF‐101‐based bio‐nanoreactor. Moreover, the immobilization by PAEA makes enzymes and NAD(P)H display excellent stability and recyclability. This study has demonstrated a facile and versatile PAEA for fabricating cofactor‐dependent multienzyme cascade nanoreactors with HOFs.