Immobilized enzyme technology has been widely used because of its advantages in enhancing enzyme stability and reusability. However, conventional methods rely on highly purified enzymes, which involve tedious extraction steps and high costs, thereby limiting their practical applications. In recent years, direct immobilization strategy based on crude enzyme has garnered attention due to its economy and high efficiency. This study developed a one-pot co-immobilization strategy where crude enzymes of recombinant cytochrome P450 reductase (rCPR), recombinant glucose dehydrogenase (rGDH), and recombinant heme oxygenase (rHO) were mixed with genipin and aminated magnetic nanoparticles to prepare magnetic cross-linked enzyme aggregates (MCLEAs) for the cascade catalysis of heme to biliverdin. After immobilization, the immobilization efficiency of rCPR, rGDH and rHO reached 64.3 %, 70.6 % and 95.2 %, respectively. The activity recovery of rCPR, rGDH, and rHO reached 105.0 %, 65.0 % and 120.0 %, respectively. The thermal and pH stability of the enzymes in MCLEAs were greatly improved, while excellent reusability and storage stability were achieved. After 7 catalytic cycles and 30 days of storage, the MCLEAs retained 50.0 % and 35.3 % of the residual activity, respectively. The cascade system significantly enhanced the catalytic performance of rHO, achieving a biliverdin production of 0.70 mu M after 10 min of reaction. This study offers a novel strategy for the simple immobilization and efficient application of complex enzyme systems.

Compared with natural enzymes, nanozymes demonstrate potential advantages in activity, stability, storability, and cost-effectiveness. Prussian blue nanoparticles (PBNPs) exhibit exceptional catalytic activity, including efficient peroxidase-like activity, due to the presence of Fe3+/Fe2+redox pairs. However, the application of PBNPs is limited by their insufficient stability, poor dispersibility, and relatively large average particle size. In this study, a novel PBNPs was prepared from Fe-BTC MOF and exhibited considerable peroxidase-like activity. Characterization revealed that the PBNPs exhibited a cubic morphology with distinct lattice fringes and an average particle size of 40 nm. The nanozymes exhibited excellent peroxidase-like catalytic activity, displaying optimal performance at pH 2.2 and a temperature of 40 degrees C along with exceptional pH and thermal stability. In addition, they showed a lower limit of detection (LOD, 1.61 mu M) and a wider linear range (2.5-20 mu M) in colorimetric detection compared with other nanozymes. This work developed a method for high density preparation of peroxidase-like PBNPs mediated by Fe-BTC MOF and offered an efficient method for H2O2 detection using the nanozyme.

Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system, exhibits multiple physiological functions. The GABA is synthesized through the decarboxylation of glutamic acid, a reaction catalyzed by the enzyme glutamate decarboxylase (GAD). In this study, magnetic nanoparticles were coated with tussah silk (TS) protein. The GAD was then immobilized via laccase-catalyzed oxidative coupling. Characterization revealed that the TS protein coated magnetic nanoparticles exhibited spherical morphology with rough surfaces, an average particle size of 27 nm, and excellent magnetic separation capability. Compared to the free GAD, the immobilized GAD exhibited significantly enhanced pH and thermal stability. After 15 days at 4 degrees C, the immobilized GAD retained 76.3 % relative activity, whereas the free enzyme showed a complete loss of activity. Furthermore, the immobilized GAD maintained approximately 50 % residual activity after seven operational recycles. This work successfully established an enzymatic crosslinking strategy for GAD immobilization on magnetic nanoparticles, providing both an efficient approach for GABA production and a novel methodology for enzyme immobilization technology.

Glutamate decarboxylase (GAD) catalyzes the conversion of L-glutamate to gamma-aminobutyric acid (GABA), a bioactive compound of therapeutic relevance. In this study, GAD was recombinantly expressed in Escherichia coli BL21 (DE3) (pET30a-GAD) and immobilized via entrapment in alginate gel with nano-calcium carbonate (nano-CaCO3) synthesized in a green deep eutectic solvent. The entrapment immobilization enhanced the nano-CaCO3 immobilized GAD catalytic properties, operational stability, and reusability. The immobilized GAD displayed optimal activity at 50 degrees C and pH 4, retained 42% activity after 3 h at 80 degrees C, and maintained 85% activity after 1 h at pH 3. The non-nano-CaCO(3 )immobilized GAD retained 21% activity after 3 h at 80 degrees C and 58% after 1 h at pH 3. The K-m and V-max of free GAD were 0.03 mM and 765.1 mu molL-1min(-1). After immobilization, the K-m and V-max of non-nano-CaCO3 were 2.85 mM and 0.30 mu mol & sdot;L-1 min(-1), and the K-m and V-max of nano-CaCO3 were 1.37 mM and 0.48 mu molL-1min(-1) respectively. Long-term storage and reusability assessments showed 52% retained activity after 40 days at 4 degrees C and over 90% after 8 cycles, compared to 27% and under 70% for the non-nano-CaCO3 immobilized GAD. The scanning electron microscopy (SEM) image confirmed the successful incorporation of nano-CaCO3 particles within the immobilized GAD beads. These results demonstrate that the nano-CaCO3 assisted alginate entrapment immobilization offers a robust and sustainable strategy for enhancing GAD stability and functionality in biocatalytic applications.

Cuttlebone (CFB) is a food waste and primarily composed of calcium carbonate. In this study, the chitosan modified CFB (chitosan@CFB) was prepared by the CFB particle coated with chitosan and then employed as an eco-friendly carrier for trypsin immobilization. The immobilized trypsin (trypsin@chitosan@CFB) was characterized by scanning electron microscopy. The optimal pH of trypsin@chitosan@CFB was 9.0 and the optimal temperature was 37 °C. The trypsin@chitosan@CFB exhibited excellent temperature, pH, storage stability, and reusability. The Km of the trypsin@chitosan@CFB was 0.64 mM when casein used as substrate. The trypsin@chitosan@CFB was used for bovine hemoglobin degradation and the result was analyzed by SDS-PAGE. The trypsin@chitosan@CFB demonstrated preferably potential for industrial applications.

Silver nanoparticles (AgNPs) are playing an increasingly important role in industries and medical fields as an antibacterial nanomaterial. Hence, green production of AgNPs is crucial. In this study, molecular simulations were employed to explore the reduction capability of D-glucose in deep eutectic solvent (DES). The AgNPs were prepared in a glucose-based DES without the addition of any reducing agents. The AgNPs were nearly spherical with diameters increasing from 32 nm to 56 nm as the silver ion concentration increased. When used the DES composed of betaine and urea as an adjuvant, the AgNPs exhibited better antibacterial activity against E. coli and B. natto compared to the AgNPs in water suspension. This research provided an effective pathway for the green production of AgNPs and proposed a new method for the production of an antibacterial agent primarily composed of AgNPs.

The waste toner in the printer poses a serious threat to the environment and human health. That is necessary to recycle toner reasonably and effectively. In this study, toner powder was mixed with acrylamide and chitosan to prepare a magnetic hydrogel adsorbent which was further used to explore the adsorption of amido black 10B dye solution. The adsorbent was characterized by SEM, FTIR, VSM and TG. The zero charge point (pHpzc) of the adsorbent was about pH 6, and the adsorption effect was better under acidic conditions. When the initial concentration of the dye solution was 200 mg/L, it reached equilibrium in about 4 h and the maximum adsorption capacity was 6.027 mg/g. When the initial concentration was 400 mg/L, it could be regarded as basic equilibrium in 6 h and the adsorption capacity was 11.106 mg/g at this time. The adsorption process well followed the pseudo-second-order kinetic model and Freundlich adsorption isotherm. The values of Delta H theta and Delta G theta indicated that the adsorption process was endothermic and spontaneous at high temperature.

This study aimed to design a Natural Deep Eutectic Solvent (NADES) for extracting capsaicin from the millet pepper (Capsicum annuum L.). Results demonstrated that NADES3 composed of choline chloride and citric acid (2 : 1 molar ratio) yielded 4.23 mg capsaicin per gram of pepper under optimized conditions (30% water content, 20 mL/g liquid-solid ratio, 20 min ultrasound). Post-enrichment with AB-8 macroporous resin, capsaicin recovery efficiency reached 87.66%. Molecular dynamics simulation revealed a stronger NADES3/capsaicin interaction compared to ethanol/capsaicin, indicating that capsaicin exhibits better homogeneous dispersion in NADES3 than in ethanol. Moreover, the recyclable NADES3 could be reused at least 4 times, showing that the proposed extraction method was eco-friendly. Thus, this study will serve as a reference for the future application of NADES in extracting other alkaloids, both in laboratory and industrial settings.

A natural deep eutectic solvent-based ultrasound-assisted simultaneous extraction (NADES-UAE) of camptothecin (CPT) and 10-hydroxycamptothecin (10-HCPT) was established. The 1.31 mg of CPT and 1.66 mg of 10-HCPT were obtained from each gram of the fruit powder of Camptotheca acuminata under the optimum conditions with a water content of 20%, a liquid-solid ratio of 12 mL/g and an ultrasound time of 20 min. The recovery efficiencies of CPT and 10-HCPT after AB-8 resin enrichment were 70.5% and 74.8%, respectively. The stronger interaction between NADES3 which was screened from 12 kinds of NADES and target components compared with methanol or water was demonstrated using molecular dynamics simulation. Moreover, the recovered NADES3 could be reused at least 4 times. The present research provided an efficient, environment-friendly, and sustainable method for extracting and recovering CPT and 10-HCPT from the fruits of C. acuminata.

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 1.10mM for the MCLEAs and 0.24mM for the MGE at 48h. This study explored a facile method for co-immobilization of (R)-PEDH and rGDH to synthesize (R)-1-(furan-2-yl) ethanol.

Laccase has a wide range of substrates and the oxidation product is water, providing an ideal choice for the biological degradation and decolorization of dyeing wastewater. This study extracted laccase from the fruiting body of Agaricus bisporus, then used o-phthalaldehyde (OPA) as a cross-linking agent and added surface-aminated Fe3O4 nanoparticles (NH2-nanoFe3O4) to prepare magnetic cross-link enzyme aggregates (MCLEAs) of laccase, which was characterized by scanning electron microscope and vibrating sample magnetometer. The optimal temperature for the catalytic reaction of the MCLEAs was 35 degrees C, and the optimal pH was 3.0. The stability to temperature pH and storage stability were all enhanced. The immobilized laccase was utilized to treat amido black 10B solution, and the decolorization rate reached 92.48%. After five cycles of use, the MCLEAs of laccase could maintain 42.91% of the relative activity and 32.31% of the relative degradation ability to amido black 10B.

Food waste cuttlebone has the potential as the raw material of carrier for enzyme immobilization. In this study, we presented a method for synthesizing CaCO3 nanoparticles coated chitosan (nano-CaCO3@chitosan) using cuttlebone as the raw material in the deep eutectic solvent of betaine and urea. The yield of nano-CaCO3@chitosan was 89.9

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.

Pancreatic ductal adenocarcinoma (PDAC) remains an extremely aggressive disease characterized by rapidly acquired multi-drug resistance, including to first-line chemotherapeutic agent gemcitabine. Autophagy is a process that is often exploited by cancer and is one of several intrinsic factors associated with resistance to gemcitabine. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of factors including Valosin-containing protein (VCP). VCP has been reported to play an important role in autophagic flux. In this study, we investigated whether the repression of VCP through miR-198 administration disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment in vitro. Moreover, we used LGA-PEI (LPNP) nanoparticles to effectively administer miR-198 to tumors in vivo, inducing tumor sensitization to gemcitabine and leading to a significant reduction in tumor burden and metastases and a concomitant downregulation of VCP expression and autophagy maturation. Our results indicate a potential therapeutic strategy for targeting gemcitabine resistant PDAC and establishes the use of LPNPs for effective therapeutic delivery of nucleic acids in vitro and in vivo.

Silk has been widely used not only in the textile field but also in non-textile applications, which is composed of inner fibrous protein, named fibroin, and outer global protein, named sericin. Due to big differences, such as appearance, solubility, amino acid composition and amount of reactive groups, silk fibroin and sericin usually need to be separated before further process. The residual sericin may influence the molecular weight, structure, morphology and properties of silk fibroin, so that degumming of silk is important and necessary, not only in textile field but also in non-textile applications. Traditional textile degumming processes, including soap, alkali or both, could bring such problems as environmental damage, heavy use of water and energy, and damage to silk fibroin. Therefore, this review aims to present a systematic work on environmentally friendly and green degumming processes of raw silk, including art of green degumming process, quantitative and qualitative evaluation, influence of degumming on molecular weight, structure, morphology and properties of silk. It is anticipated that rational selection and design of environmentally friendly and green degumming process is quite important and meaningful, not only for textile application but also for non-textile application.

Nanotechnology has become the most promising domain to boost the efficiency of enzymes. Enzymes are vital as a green catalyst in many industries, food, pharmaceutical and biomedical, etc. The immobilization process of the enzyme increases its catalytic properties. In this research, a novel method is presented to describe the effect of nano-calcium carbonate on the characteristics of immobilized β-glucosidase, which was extracted from the Agrocybe aegirit. The nano-CaCO3 was produced using the eco-friendly natural deep eutectic solvent. The pure nano -CaCO3 was observed as vaterite, with a size of about 300 nm. The nano-calcium carbonate was coated by a natural polymer sodium alginate compound and then adsorbed chitosan. Further, this obtained composite is cross-linked by the bioactive genipin to immobilize the β-glucosidase. The enzyme/protein loading ratio to the supports was 1:4, respectively. The recovery efficiency of immobilized β-glucosidase was 89.3%, and immobilization yield was 96.452%. Chitosan-coated nano-CaCO3 was used as a carrier for immobilization of β-glucosidase to improve its stability and reusability. In addition to stability and reusability, pH tolerance, temperature tolerance, and enzyme kinetics are the significant parameters that illustrate the proficiency of an immobilized enzyme. The measured optimal enzymatic reaction conditions for the immobilized β-glucosidase were 50 °C and pH 6. Furthermore, it has shown noticeable improvements in thermo-stability and pH tolerance. Temperature tolerance was observed 50% to the initial activity of the immobilized enzyme even after the 3 h of incubation at 50 °C, while pH tolerance was noticed more than 50% and 40% at pH 7 and 8, respectively. The Km and Vmax values of free and immobilized β-glucosidase to 4-nitrophenyl β-D-glucopyranoside were 1.549 μmol/L/min, 0.346 mmol/L and 0.532 μmol/L/min, 0.080 mmol/L, respectively. The immobilized β-glucosidase retains its storability 80% even after 30 days of storage at 4 °C and maintains 93.1% of its residual activity by reusing up to 10 cycles.

Enzyme immobilization provides ideal operating conditions for enzymes stabilization and sustainable recycling. In this work, as a kind of clay material, montmorillonite (MTL) was chosen for immobilizing the β-glucosidase extracted from Agrocybe aegirit. The immobilized β-glucosidase via partly cross-linking enzyme aggregates (pCLEAs) formed by self-catalysis provided biocatalysts with satisfactory thermal and pH stability. Compared to the glutaraldehyde cross-linked, the immobilized β-glucosidase (β-G-pCLEAs@MTL) exhibited significantly higher immobilization efficiency (IE) and immobilization yield (IY), which were 80.6% and 76.9%, respectively. The β-G-pCLEAs@MTL also showed better stability and preferable reusability. And the activity of the β-G-pCLEAs@MTL remained 85.0% after 5 cycles and 74.7% after 10 cycles. Therefore, the method based on the pre- crosslinking to form pCLEAs and after-immobilization can effectively improve IY and IE. In addition, MTL seems to be a good alternative carrier to immobilize other enzymes for industrial application.

The search for seeking novel immobilized enzyme carriers is a hot topic in the field of enzyme engineering. As a kind of common office consumable, magnetic printing toner has been terribly wasted, leading to potential harm to the environment. We try to use printing toner as carrier for enzyme immobilization to realize waste reuse. In this paper, the laccase extracted from Agaricus bisporus was successfully immobilized on chitosan coated magnetic printing toner by glutaraldehyde linking. The use of printing toner is proved to be feasible. The results show that the optimal temperature of immobilized laccase is 40 degrees C, and the optimal pH is 3.0. Compared with free laccase, the optimal temperature and optimal pH have been obviously changed after immobilization. And the pH tolerance has been significantly improved, which can remain 53% activity after 5 cycles. Because of the use of magnetic printing toner, it is more convenient to recycle the immobilized enzyme. In this study, printing toner was used as carrier for the immobilization of enzyme and the discarded printing toner recycling was realized.

Coenzymes are required in the reaction catalyzed by oxidoreductases. The research on coenzyme regeneration has always been a hot topic in the field of biological catalysis. In this work, we tried to construct a coimmobilization system of glucose dehydrogenase (GDH), (R)-1-phenylethanol dehydrogenase ((R)-PEDH) and coenzyme with functional magnetic nanoparticles as carriers, so as to realize chiral (R)-1-phenylethanol highly selective production and coenzyme effective recycling. The enzymatic properties of immobilized enzymes were explored. The results showed that the immobilized enzymes had improved stability and fine reusability. In addition, during the catalytic coupling reaction at a final concentration of 2.63 mu M NAD+, the total turnover number (TTN) was as high as 924, and the volumetric productivity of (R)-1-phenylethanol was 37.06 mg.L-1.h(-1). After reused for 10 cycles, the catalytic efficiency of immobilized coenzyme can still be maintained at more than 30%. From multiple perspectives, the immobilized dual-enzymes and coenzyme system seems to be a multifunctional system for (R)-1-phenylethanol production and coenzyme regeneration.

Abstract As a new environmentally friendly and sustainable green solvent, deep eutectic solvent (DES) was considered to be the most suitable alternative solvent for ionic liquids, with wide application. In this study, we reported a novel method of the synthesis of Fe3O4 nanoparticles (Nano-Fe3O4) in the natural deep eutectic solvent (NADES) of betaine/urea. Compared with traditional synthetic methods, this method of synthesising magnetic nanoparticles with the NADES as solvent possesses the superiority of mild reaction conditions, no need for nitrogen protection and short time-consuming. The yield of Nano-Fe3O4 synthesised in the NADES was as high as 94.9%. The synthesised Nano-Fe3O4 were coated with silicon and used to immobilise β-glucosidase extracted from Agrocybe aegerit. The results showed that the immobilisation efficiency of the β-glucosidase was 87.5% and the immobilisation yield was 86.2%. The optimum reaction conditions of the immobilised β-glucosidase were 55 °C and pH 6.0. In addition, the thermostability, pH stability and storage stability of the β-glucosidase were greatly improved after its immobilisation on the Nano-Fe3O4. After reused for 10 cycles, the residual activity of the immobilised β-glucosidase was still more than 72.4%. From multiple perspectives, the synthesised Nano-Fe3O4 in the NADES seems to be an excellent carrier for enzyme immobilisation.